We thank Timothy F. Sharbel (IPK Gatersleben) for providing Boechera divaricarpa seeds and Sharon Kessler (University of Oklahoma) for critical reading and proofreading. Work on cell type-specific transcriptome analyses to study gametophyte development and apomixis in UG&#180;s laboratory is supported by the University of Z&#252;rich, by a fellowship of the "Deutsche Forschungsgemeinschaft" and the Marie Curie project IDEAGENA to AS, by grants from the "Staatssekretariat f&#252;r Bildung und Forschung" in the framework of COST action FA0903 (to UG and AS) and the Swiss National Foundation (to UG).

Note: This protocol describes tissue preparation, laser assisted microdissection, and RNA extraction for transcriptional profiling. Always use gloves throughout all steps of the protocol. Study and consider the safety instructions for each chemical used. In particular, keep in mind that Xylol is harmful and can penetrate gloves and that methanol is toxic. For all instruments used, please refer to user manuals accordingly.
1. Removal of RNAse Activity from Glassware and Other Equipment
<ol>
	<li>Prior to use, wrap any glassware&#160; in aluminum foil prior to baking for ~ 8 hr at 180 &#176;C to ensure RNAse free quality.</li>
	<li>Treat any metal surfaces (e.g., pair of tweezers) as well as the work bench and the heating plate with an appropriate RNAse decontamination solution. Afterwards, wash the surfaces with RNAse-free water to remove the decontamination solution. Subsequently, clean the surfaces further with 70% EtOH.</li>
	<li>Use all plastic consumables (e.g., tubes) brand-new without any pretreatment. If available, use plastic consumables that are certified to be RNAse free.</li>
</ol>
2. Tissue Fixation
<ol>
	<li>Prepare 10 ml of farmer&#39;s fixative (3:1; v/v ethanol:acetic acid) in a fresh 15 ml tube on ice. (Always prepare fresh Farmer&#39;s fixative before use). As an alternative, the non-crosslinking fixative ethanol:acetic acid (5:1; v/v) can be used 18.</li>
	<li>Use a fine pair of tweezers to collect buds at the developmental stage of interest. Immediately submerge buds in the ice-cold fixative. Use 10 ml of fixative for the fixation of ~ 20 buds or flowers from Arabidopsis or Boechera. Note: When using plant species with larger flowers it is recommended to dissect the flowers with a razor blade to generate smaller pieces prior to fixation.
	<ol>
		<li>In order to obtain mature gametophytes, emasculate the 2 - 3 largest flower buds of the inflorescence 2 days prior to harvest.</li>
	</ol>
	</li>
	<li>Fill the bottom of an exsiccator with ice. Open the tubes containing the samples, e.g., flowers, and place them on ice, either by directly putting them into the ice in a way that they cannot fall over or by using an appropriate holder. Vacuum infiltrate for 15 min prior to release of the vacuum.
	<ol>
		<li>Repeat the vacuum infiltration once for 15 min.</li>
	</ol>
	</li>
	<li>Release the vacuum and store overnight (~ 12 hr) on ice. Cover the ice bucket with a lid or aluminum foil and store the bucket in a 4 &#176;C room or refrigerator in order to prevent the ice from thawing. Note: Prolonging the fixation time is not recommended.</li>
</ol>
3. Tissue Embedding, Thin Sectioning, and Mounting on Slides for LAM
<ol>
	<li>Tissue embedding.
	<ol>
		<li>Exchange the fixative to 70% ethanol prepared with pure ethanol and RNAse free water. Leave the samples on ice until further processing. Start embedding of the samples the same day. In case no tissue-processing machine is available, proceed to manual embedding as described elsewhere 9,11 and continue with step 3.1.11.</li>
		<li>Gently transfer the samples to tissue embedding cassettes with a pair of tweezers. Firmly close the cassettes. CRITICAL STEP: Avoid even partial drying of the sample during this step. Avoid squeezing of samples with the tweezers. 
		Note: To label the cassettes a pencil should be used, ideally by writing on the inclined surface of the cassette.</li>
		<li>Store the embedding cassette loaded with the buds or flowers in 70% ethanol until all material is transferred to cassettes and the embedding machine can be loaded. For this purpose, use a glass staining trough filled with 70% ethanol.</li>
		<li>Remove the basket of the tissue processor from the retort of the machine and transfer the embedding cassettes to the basket with the inclined surfaces facing the top and in the same direction. CRITICAL STEP: Perform this quickly to prevent any drying of the tissues. If processing many embedding cassettes at once, place the basket into an appropriate RNAse free container filled with 70% ethanol prior to loading the samples.</li>
		<li>Put the lid on the basket and put the basket back into the retort. Close the retort. 
		Note: The tissue processor automatically dehydrates the samples, changes the solvent to Xylol, and does the infiltration with melted paraffin wax. Typically, this is done overnight.</li>
		<li>Start the program of the tissue processor. Recommended standard settings are 1 hr 70% EtOH, three times 1 hr 90% EtOH, three times 1 hr 100% EtOH, two times 1 hr 100% Xylol, one time 1 hr 15 min 100% Xylol at room temperature, followed by infiltration with paraffin wax two times for 1 hr and one time for 3 hr at 56 &#176;C 11. 
		Note: To ensure that the blocking station is ready with the wax melted, switch the machine on several hours before start or use the timer function to select the approximate starting time. 
		Note: In case the tissue cannot be processed immediately afterwards, longer incubation times in paraffin wax at 56 &#176;C are possible instead of the 3 hr at 56 &#176;C. Refer to the user manual of the instrument. Typically, the cassettes with tissues will automatically remain in melted wax until the &#34;empty retort&#34; command is approved. If the wax is not melted when starting the tissue processor, the retort is filled with 70% ethanol and the program remains on hold until ready to start.</li>
		<li>Empty the retort and immediately transfer the samples to a paraffin wax bath at 56 &#176;C in a blocking station. Remove the lid from the basket and cover the paraffin bath of the blocking station with its lid.</li>
		<li>Pile as many fresh balancing trays on the surface of the blocking station heated to 56 &#176;C as there are cassettes in the basket. 
		Note: An ethanol-resistant permanent marker can be used to label the bottom of the balancing trays. Acetone-resistant permanent markers are not recommended.</li>
		<li>Using the blocking station, fill a pre-warmed fresh plastic balancing tray with melted paraffin wax at 56 &#176;C. Lift the lid of the paraffin bath, pick up one cassette only at a time and transfer the samples from the cassettes to the liquid paraffin wax at 56 &#176;C in the balancing tray using a pair of tweezers. 
		CRITICAL STEP: Avoid any hardening of the wax surrounding the sample while handling the cassette by working quickly.
		<ol>
			<li>Position all samples according to the needs using a preparation needle before hardening of the wax. Typically, several flowers are arranged in a balancing tray individually spaced about 1 cm in each direction.</li>
			<li>Repeat step 3.1.9 until all samples are transferred.</li>
		</ol>
		</li>
		<li>Place the basket back to the tissue processor and clean the retort using an appropriate program (refer to user manual).</li>
		<li>Switch off tissue processor and blocking station. Continue with step 3.1.13.</li>
		<li>In case no tissue infiltration machine and no embedding station is available, use a heating oven to melt paraffin wax at 56 &#176;C. On the bench, fill the melted wax in plastic balancing trays, transfer the samples to the wax, and use preparation needles to position the samples.</li>
		<li>Allow the paraffin wax to harden at room temperature for ~ 1 - 3 hr before storing the samples at 4 &#176;C. Samples can subsequently be processed freshly or stored for up to several months.</li>
	</ol>
	</li>
	<li>Preparation of thin sections and slides for LAM.
	<ol>
		<li>On a light table illuminating the wax block from below, remove the paraffin wax with the samples from the surrounding plastic trays. Dissect out a squared wax block containing the tissue, e.g., a bud or flower, using an RNAse free razor blade. To this aim, always orient the samples towards the top of the wax block (also see Figure 1).
		<ol>
			<li>Prepare wax blocks of all samples that should be used for LAM at a time.</li>
		</ol>
		</li>
		<li>Mount the blocks to process embedding cassettes filled with hardened paraffin wax: melt a small piece or droplet of paraffin wax on the tip of an appropriate spatula using an Ethanol lamp and use the hot wax to fix the block on the support (with the embedded tissues on top).
		<ol>
			<li>Allow the wax to harden for at least 20 min at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Remove surplus wax surrounding the sample with a razor blade on the light table. Make sure to keep a square surface with parallel edges (see Figure 1).</li>
		<li>Set the heating plate to 42 &#176;C.</li>
		<li>Safely mount the samples to the microtome and prepare 6 - 10 &#181;m thick sections. Refer to the manufacturer&#39;s instructions for using the microtome. Note: While thinner sections will often give better structural resolution, larger amount of RNA of higher quality can be obtained with thicker sections. For the cells of Boechera mature gametophytes we use 7 &#181;m as the default. Moving rather slowly with the samples over the microtome knife often results in better histology.</li>
		<li>Always transfer the paraffin ribbons at a length of approximately 10 - 15 cm into a plastic box with a black cardboard at the bottom before positioning them in LAM slides.</li>
		<li>If possible, pre-select the parts of the paraffin strips containing the cell- or tissue types of interest, e.g., ovules, under a dissecting-scope and discard the rest.</li>
	</ol>
	</li>
	<li>Preparation of the slides for LAM.
	<ol>
		<li>Place the required amount of metal-framed slides (typically 5 - 10) with the flat surface on top on the heating plate.</li>
		<li>Pipet ~ 1 - 2 ml of RNAse free water on the plastic part of the slide. Using a razor blade, cut the paraffin ribbons in short pieces of about 4 - 5 cm long enough to span the plastic windows. Use a pair of tweezers and a preparation needle to ideally place the paraffin strips parallel to each other on the plastic windows of the slides. Carefully lift the slide at one end to remove the water and place the slides back.
		<ol>
			<li>Alternatively, place the heating plate under a chemical hood. Apply a small volume of methanol to the plastic part of the slide just sufficient to wet the surface (this can cause a slight corrugation of the slides). Place the paraffin strips on the slides. 
			Note: For reasons of toxicity, avoid use of methanol if possible. However, the use of methanol at this step does improve the RNA quality in case the quality achieved by mounting on water is not sufficient. It is worthwhile testing these alternatives at the start of a new project, as the RNA quality achieved varies with cell type and species under investigation.</li>
		</ol>
		</li>
		<li>Dry the slides overnight on the heating plate at 42 &#176;C for ~ 12 hr. Cover the heating plate with a plastic lid that does not touch the slides. Ensure that the plastic lid does not prevent the exchange of air. To this aim the lid can be placed on pipet boxes or similar to be lifted.
		<ol>
			<li>Alternatively, reduce the drying time to a minimum of two hours or until the tissue appears totally dry. The reduction of time from slicing to collection may improve RNA quality.</li>
		</ol>
		</li>
		<li>Under a chemical hood, de-wax the slides 2 times for 10 min in Xylol using appropriate slide holders. Make sure to fully submerge the plastic part of each slide only but to allow removal of the slide by touching the broader end of the metal frame that is kept dry.</li>
		<li>Allow the slides to dry for ~ 10 min under the chemical hood prior to LAM. 
		Note: Slides not immediately processed can be placed back on the heating plate at 42 &#176;C with the tissue facing up for up to a few hours. This will prevent any humidity from compromising RNA quality until further processing.</li>
	</ol>
	</li>
</ol>
4. Laser-assisted Microdissection (LAM)
Note: The procedure for LAM will vary with the instrument used. Certain details of this protocol are adapted to a specific instrument and the technology of collecting the samples on a cap making use of electrostatic forces. In addition, details might vary even between different versions of the software. Please refer to manufacturer&#39;s instructions and user handbooks for a detailed description and specific instructions on instrument and software.
<ol>
	<li>Switch on the devices for LAM (computer, microscope, laser control box).</li>
	<li>Start the program steering the microscope and laser.</li>
	<li>Switch on the laser by pressing the corresponding button on the control box.</li>
	<li>Place a cap (0.5 ml, without diffusor) into the cap holder and position it in the instrument.</li>
	<li>Support the LAM slide with a glass slide for microscopy. Ensure that the flat surface of the slide with the tissue attached faces the glass slide.</li>
	<li>Place the slide in the instrument with the glass slide underneath.</li>
	<li>Select the 4X Objective. In the program, select to move the cap to the &#34;up&#34; position and proceed to make a scan of the slide.</li>
	<li>Search through the slide and identify the structures of interest. Pinpoint and save their positions on the slide to be able to move back to the exact position for the cutting step.</li>
	<li>Select the appropriate objective (e.g. 40X or 60X).</li>
	<li>If needed, adjust laser speed, focus, and laser power, and also calibrate the stage movement by referring to the user manual of the instrument. Once an account is set up for the specific tissue of interest, the settings can be reused.</li>
	<li>Verify the laser position by a single shot by pressing the &#34;laser shot&#34; button of the program, ideally in a part of the membrane without tissue.
	<ol>
		<li>If required, calibrate the laser position by following the manufacturer&#39;s instructions for the instrument.</li>
	</ol>
	</li>
	<li>Verify the calibration of the objective by moving an obvious structure to all four corners of the software window on the monitor, keeping the right mouse button pressed. Check that the position of the hand with respect to the obvious structure is the same in all four corners. Otherwise calibrate the objectives following the software manual.</li>
	<li>With the cap in the &#39;down&#39; position, use the &#39;hand-pen&#39; tool to mark the borders of the cell type or tissue of interest, e.g., the egg cell. Dissect the cell of interest with the laser by pressing &#39;cut&#39;. Note: Often, sectioning needs to be repeated if the border around the cell was not entirely dissected at once. In this case, it is recommended to set the software to automatically do two repeats of the section as a standard.</li>
	<li>Lift the cap and move to the next saved position with the structure of interest. Repeat step 4.13 of the procedure to dissect the next cell sections until all cells of interest from one slide are collected. Make sure the cap is positioned in a way that the new section sticks to an empty position on the cap. 
	Note: It is recommended to isolate larger pieces of tissues (e.g., sections of whole flowers or parts of it) from each slide after the isolation of single cell sections on a fresh cap. These can be used for RNA quality control.</li>
	<li>Remove the slide and continue with the next slide by repeating steps 4.4 - 4.9 and 4.13 to 4.14.</li>
	<li>Repeat step 4.15 and related steps until the cell types of interest from all slides have been isolated. Note: It is not recommended to harvest for longer than ~ 4 - 5 hr on the same cap.</li>
	<li>Visually inspect the cap surface after LAM to exclude unspecific contaminations of the sample. While the non-dissected tissues are protected with the foil, dust can stick to the surface due to electrostatic adhesion.
	<ol>
		<li>To visually inspect the cap, remove the slide from the instrument. Place the cap holder in the &#39;down&#39; position and inspect the surface with the 4X objective.</li>
		<li>In case small pieces of dust are visible, remove them with a small (2 &#181;l) pipette tip. Alternatively, mount the slide back on the instrument, place the cap on a free part of the slide (no tissues), and completely destroy the contamination using the laser.</li>
	</ol>
	</li>
	<li>Close caps and freeze the dry sections at -80 &#176;C until further use. If required, samples can be stored for up to several weeks. However, proceeding quickly at this step is recommended.</li>
</ol>
5. RNA Isolation and Quality Control
Note: RNA can be isolated by any method suitable for small amounts of RNA. In this protocol the use of an RNA isolation kit specified for small amounts of RNA is described. Follow the manufacturer&#39;s instructions.
<ol>
	<li>Use the tubes with the sample attached to the cap either directly or after removal from -80 &#176;C.
	<ol>
		<li>Open the tubes and carefully pipet 11 &#181;l of extraction buffer to the wall of each tube using filter tips. Change tips between samples.</li>
		<li>Close the lid and shake the extraction buffer down to the cap of the tube.</li>
		<li>Place the tubes with the cap down in a heating oven preheated to 42 &#176;C. Incubate for 30 min following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Follow the manufacturer&#39;s instructions for column preparation and collecting the extract at the bottom of the tubes.
	<ol>
		<li>If material from more than one cap needs to be combined into one sample, proceed with steps 5.1.1 - 5.1.2 individually for each tube/cap.</li>
		<li>Afterwards, pool the extracts for one sample in one tube and measure the amount of pooled extract with a pipette prior to addition of an equivalent amount of EtOH (see manufacturer&#39;s instructions).</li>
		<li>Ensure that the amount of EtOH added before loading the column is equal to the volume of the pooled extract.</li>
		<li>After RNA precipitation with EtOH, proceed quickly to the loading of the columns following manufacturer&#39;s instructions. Continue the protocol with the 100 x g centrifugation step.</li>
		<li>After the contents of each collecting tube have been passed through the column, perform a 16,000 x g centrifugation step for 30 sec to remove the flow-through following manufacturer&#39;s instructions.</li>
		<li>Continue with RNA extraction and purification following manufacturer&#39;s instructions.</li>
		<li>After loading of the elution buffer to the column allow the column to incubate for 1 min. Continue to load the tubes into the centrifuge following the instructions in the manufacturer&#39;s instructions. Note: An additional centrifugation step for 1 min at 100 x g prior to elution as indicated in the protocol can increase the efficiency of elution.</li>
	</ol>
	</li>
	<li>Extract RNA from the controls (larger tissue sections) for RNA quality control following steps 5.1 to 5.2.7 of this protocol.</li>
	<li>For RNA quality control load 1 &#181;l undiluted total RNA of each control sample on the Bioanalyzer using an RNA Pico Chip following the manufacturer&#39;s instructions. 
	Note: The extracted RNA can be used for linear amplification and transcriptome analysis using microarrays or RNA-Seq 7,11,13,14. Ideally, the quality control should indicate an RNA Integrity Number (RIN) of at least 7. A number of suppliers provide suitable kits for amplification, suitable examples are given in 9.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The Protocol is Suitable for Different Cell and Tissue Types
LAM combined with transcriptome analyses by microarrays or RNA-Seq is a valuable tool to gain insights into the specific patterns of gene activity regulating developmental or physiological processes 7-11,13,14. However, the suitability of this method for any given cell type is critically dependent on structural issues. The cell needs to be clearly visible and unambiguously identifiable in the dry sections ...

In transcriptional studies done at the tissue level, the transcriptomes of highly specialized but rare cell types are often masked by the more abundant surrounding cells. An example for such highly specialized cell types are the cells of the female reproductive lineage (germline) in plants. The female germline is specified within developing ovules, the precursors of seeds inside the gynoecium of the flower 1,2. The megaspore mother cell (MMC) is the first cell of the female germline. It undergoes meiosis to form a tetrad of reduced megaspores. Typically, only one of these megaspores survives and divides mitotically without cytokinesis, i.e., in a syncytium. These mitoses are followed by cellularization to form the mature gametophyte, which typically consists of four cell types: three antipodals, two synergid cells, the egg, and the central cell. The egg and central cells are the female gametes that get fertilized by two sperm cells during double fertilization to give rise to the embryo and endosperm of the developing seed 1,2. In the sexual model system Arabidopsis thaliana, only ~ 50 seeds develop per flower while about 50 - 80 seeds develop per flower in the closely related genus Boechera. Thus, the female germline consists of only a few highly specialized cell types, making it an excellent model to study developmental processes, such as cell specification and differentiation.
Moreover, insights into the gene regulatory processes governing plant reproduction can be of applied value. In plants, both sexual and asexual reproduction through seeds (apomixis) can occur. While sexual reproduction generates genetic diversity in a population, apomixis leads to the formation of clonal offspring that is genetically identical to the mother plant. Therefore, apomixis has great potential for applications in agriculture and seed production, as even complex maternal genotypes can be maintained unaltered over several generations 3,4,5. Because apomixis does not naturally occur in any major crop species, the engineering of apomixis in crops is of great interest 3,4,5. However, this long-term goal is difficult to achieve because the underlying genetic and molecular basis of apomixis is not understood in sufficient detail 6.
To gain insights into the transcriptional basis governing apomictic reproduction, cell type-specific transcriptional profiling using laser-assisted microdissection (LAM) and next generation sequencing (NGS) represents a very powerful approach 7,8. LAM has first been established for animal and biomedical research. In the past few years LAM has also been applied to plant biology 6,9,10. In contrast to other methods allowing profiling of individual cell and tissue types, LAM does not require the generation of marker lines 6,9,10. Therefore, it can be applied to any cell or tissue type without prior molecular knowledge. Another advantage of LAM is that it can be applied to any cell type as long as the cell can be recognized in dry sections based on position and/or structural features. LAM has the additional advantage that fixed tissues are used, which prevents changes of the transcriptional profile during processing.
The tissue of interest, e.g., floral tissue, is fixed in a non-crosslinking fixative prior to embedding in paraffin wax. Embedding in paraffin wax can be done manually, following established protocols 9,11. However, the use of an automated tissue processor for dehydration and infiltration with the wax generally results in higher reproducibility in terms of the conservation of RNA quality and tissue morphology. The alternative strategy of embedding tissues in resin has also been successfully used for cell type-specific analyses by LAM 8. However, the use of an automated tissue processor for embedding in wax is very time efficient, as many samples can be processed at once requiring a minimum of hands-on time. While typically no significant loss of RNA quality occurs during fixation and embedding, the preparation of thin sections with the microtome and, in particular, the mounting on the frameslides used for LAM remains a critical step for preservation of RNA quality. This has previously been noted and the use of a tape transfer system has been described to result in better RNA quality at this step 12. However, this adds an additional time-consuming step during preparation of the slides and also requires special equipment. The optimized protocol described below reproducibly produces RNA that is of sufficient quality for transcriptional profiling with GeneCHIPs and Next Generation Sequencing (NGS) approaches 7,11,13,14. In addition, with the laser microdissection microscope used, a high purity of the isolated cell types is routinely produced 7,11,13,14.
The genus Boechera is an excellent model system for studying the key steps of apomictic reproduction. In Boechera, a variety of different sexual and apomictic accessions have been identified and can be used for comparative analyses 15,16,17. In a comparison of cell type-specific transcriptomes of cells from the female germline from sexual Arabidopsis and apomictic Boechera, we identified genes and pathways that are differentially expressed, thereby identifying new aspects of the regulatory processes governing apomixis 7. In addition, this study verified the suitability of LAM for cell type-specific transcriptional analyses of small and rare cell types. We have already used this protocol for the analysis of different cell types in a variety of plant species, but species- and tissue-specific modifications to the protocol may be required in certain cases.

<table><tbody><tr><td>Ethanol</td><td>VWR</td><td>1,009,861,000</td><td>absolute EMPROVE Ph Eur,BP,USP</td></tr><tr><td>15 ml falcon centrifuge tubes</td><td>VWR</td><td>62406-200</td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent</td><td>G2939AA</td><td></td></tr><tr><td>Acetic Acid</td><td>Applichem</td><td>A3686,2500</td><td>100% Molecular biology grade</td></tr><tr><td>Ambion Nuclease free water</td><td>life technologies</td><td>AM9932</td><td></td></tr><tr><td>ASP200 S</td><td>Leica</td><td>14048043624</td><td>tissue processor&amp;nbsp;</td></tr><tr><td>black cardboard</td><td></td><td></td><td>can be purchased in special paper shops</td></tr><tr><td>DNA- and RNAse-free Frame Slides</td><td>Micro Dissect GmbH</td><td></td><td>1, 4 &amp;micro;m PET-membrane; can also be purchased from Leica</td></tr><tr><td>Dumond Forceps</td><td>Actimed</td><td>0208-5SPSF-PS</td><td></td></tr><tr><td>ethanol lamp</td><td></td><td></td><td></td></tr><tr><td>exsiccator</td><td>Sigma-Aldrich</td><td>Z354074-1EA</td><td>Nalgene Vaccuum Dessicator or similar equipment</td></tr><tr><td>filter tips 10&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60010</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 1,000&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60010</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 20&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60020</td><td>can be replaced by similar tips</td></tr><tr><td>filter tips 200&amp;nbsp;&amp;micro;l</td><td>Axon Lab AG</td><td>AL60200</td><td>can be replaced by similar tips</td></tr><tr><td>forceps precision</td><td>VWE</td><td>232-1221</td><td></td></tr><tr><td>glass slide holder</td><td>Huber &amp;amp; Co.AG</td><td>10.0540.01</td><td>F&amp;auml;rbek&amp;auml;sten nach Hellendahl</td></tr><tr><td>glass staining trough</td><td>Huber &amp;amp; Co.AG</td><td>10.0570.01</td><td>F&amp;auml;rbekasten</td></tr><tr><td>Heated Paraffin Embedding Module Leica</td><td>Leica</td><td>Leica EG 1150 H</td><td>blocking station, similar devises are suitable</td></tr><tr><td>Heating and Drying Table</td><td>Medax</td><td>15501</td><td>other models and/or suppliers are suitable</td></tr><tr><td>ice bucket</td><td>VWR ice bucket with lid&amp;nbsp;</td><td>10146-184</td><td>similar buckets equally suitable</td></tr><tr><td>light table</td><td>UVP An Analytical Jena Company</td><td>TW-26&amp;nbsp;</td><td>white light transluminator</td></tr><tr><td>microscope slide</td><td>Thermo Scientific</td><td>10143562CE</td><td>cut edges</td></tr><tr><td>microtome blade</td><td>Thermo Scientific</td><td>FEAHS35</td><td>S35 microtome blade disposable</td></tr><tr><td>MMI Cell Cut Plus Instrument</td><td>MMI (Molecular Machines and Industries)</td><td></td><td></td></tr><tr><td>Non-stick, RNAse free Microfuge tubes, 2 ml</td><td>life technologies</td><td>AM12475</td><td></td></tr><tr><td>Paraplast X-TRA</td><td>Roth</td><td>X882.2</td><td>for histology</td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>life technologies</td><td>KIT0204</td><td>Arcturus PicoPure RNA Isolation Kit</td></tr><tr><td>plastic balancing trays</td><td>Semadeni AG</td><td>2513</td><td></td></tr><tr><td>plastic box</td><td>Semadeni AG</td><td>2971</td><td>Plastikdose PS</td></tr><tr><td>plastic lid for heating plate</td><td></td><td></td><td>homemade</td></tr><tr><td>preparation needle</td><td>VWR</td><td>631-7159</td><td></td></tr><tr><td>RNA 6000 Pico Kit</td><td>Agilent</td><td>5067-1513</td><td></td></tr><tr><td>RNAse free microfuge tubes</td><td>life technologies</td><td>AM12400</td><td></td></tr><tr><td>RNAse ZAP Decontamination Solution</td><td>life technologies</td><td>AM9780</td><td></td></tr><tr><td>Semi-automated Rotary Microtome&amp;nbsp;</td><td>Leica</td><td>RM2245</td><td>similar devices are equally suitable</td></tr><tr><td>Tissue Loc Histo Screen Cassettes</td><td>Thermo Scientific</td><td>C-1000_AQ</td><td>similar cassettes of other suppliers are suitable</td></tr><tr><td>Tubes with adhesive lid, without diffusor 500 &amp;micro;l&amp;nbsp;</td><td>MMI (Molecular Machines and Industries)</td><td>50204</td><td></td></tr><tr><td>Xylol (Isomere) ROTIPURAN</td><td>VWR</td><td>4436.2</td><td>min. 99%, p.a., ACS, ISO SP</td></tr><tr><td>process embedding cassettes</td><td>Leica</td><td>14039440000</td><td>Leica Jet Cassette I without lid</td></tr><tr><td>Universal Oven</td><td>Memmert</td><td>UF55</td><td>other models and/or suppliers are suitable</td></tr></tbody></table>

laser-assisted microdissection (lam) as a tool for transcriptional profiling of individual cell types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Sample Preparation and LAM are done in Consecutive Steps
A number of consecutive steps are required to prepare RNA for transcriptional analysis from selected cell types by LAM (Figure 1). This starts with the harvest of the flowers and immediate fixation to ensure that the RNA population remains unchanged after harvest. The tissue is embedded, sectioned, and mounted on slides. This allows the is...

Watch this Scientific Journal Video about Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types at JoVE.com

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

laser-assisted-microdissection-lam-as-tool-for-transcriptional

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.2K Views.  University of Zürich & Zürich-Basel Plant Science Center. Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera. 

Video: Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular ...

Genetics

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Developmental Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Medicine

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

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Laser Capture Microdissection of Mammalian Tissue

Transcriptome Analysis of Single Cells

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser Microdissection for Species-Agnostic Single-Tissue Applications