Name: the cutting and floating method for paraffin-embedded tissue for sectioning
Uploaded: 2018-09-05T15:30:00.000+00:00
Duration: 5 min
Description: Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

Questo lavoro è stato supportato dal National Natural Science Foundation della Cina (Grant n. 31400936, 31460260) e il Natural Science Foundation di Jiangxi Provincia della Cina (20171BAB215020). Ringraziamo anche il programma congiunto tra Università di Nanchang e Queen Mary University of London per sostenere questo lavoro.

Tutti i metodi descritti qui sono stati approvati dalla cura degli animali e uso Comitato di Università di Nanchang.
1. Montare l'attrezzatura e collegare il microtomo
<ol><li>Progettare il parametro per i requisiti (integrativa Figura 1).</li>
	<li>Inviare il parametro per una fabbrica locale per produrre i pannelli acrilici.</li>
	<li>Montare tutte le parti in sequenza: utilizzare cloroformio per combinare 7 pannelli acrilici commerciale in un serbatoio con un bagno di acqua e canale di sezione (Figura 1). Attenzione: Cloroformio produce sostanze tossiche quando incontra la luce e l'ossigeno. Montare l'apparecchiatura in una cappa aspirante.</li>
	<li>Installare l'elemento riscaldante elettrico tubolare attraverso il foro nel bordo acrilico #1 (Figura 2A, punta di freccia).</li>
	<li>Per installare il regolatore di temperatura, creare un piccolo foro sul bordo acrilico #2 con un trapano elettrico e installare il rilevatore di temperatura (Figura 2A, freccia). Installare un termostato digitale sul serbatoio (Figura 2A). Nota: Il rilevatore di temperatura dovrebbe essere all'interno di 1 cm sotto la superficie dell'acqua.</li>
	<li>Per fornire l'accesso di potere, regolare la tensione sotto 24 V prima di accendere l'apparecchio.</li>
	<li>Rimuovere il vassoio dei rifiuti di sezione dal microtomo.</li>
	<li>Collegare il canale di sezioni con il supporto della lama del microtomo con sigillante siliconico neutro (Figura 2B). Nota: Non striscio qualsiasi sigillante siliconico neutro sopra la parte superiore della lama, affinché la vecchia lama può essere facilmente sostituita.</li>
	<li>A secco durante la notte per consentire il cloroformio e neutro del silicone sigillante per solidificare.</li>
	<li>Installare la lama per il supporto della lama. Versare acqua nel bagno d'acqua (Figura 2). La superficie dell'acqua deve solo toccare la parte superiore della lama (Figura 2D, testa di freccia).</li>
</ol>2. paraffina sezionamento
Nota: Detriti spesso si accumulano sui bordi superiori o inferiori del blocco di cera e la superficie dell'acqua. Questo detriti deve essere deselezionato regolarmente.
<ol><li>
Eseguire paraffina convenzionale sezionamento10.
	<ol>
<li>Bloccare il preparato di paraffina-incastonato su un microtomo rotativo e posizionare la lama in un portalama.</li>
		<li>Ruotare il volantino e la sezione del campione.</li>
		<li>Trasferire il nastro sezionato a bagno d'acqua calda di 38,5 ° C.</li>
		<li>Raccogliere che le sezioni sul microscopio scivolare dalla superficie dell'acqua dopo lo svolgimento.</li>
		<li>Asciugare i vetrini in un forno a 42 ° C durante la notte.</li>
	</ol>
</li>
	<li>
Paraffina migliorata sezionamento (Figura 3)
	<ol>
<li>Accendere l'apparecchiatura.</li>
		<li>Aprire la stufa con interruttore di rilevamento temperatura e impostare la temperatura mirata tra 38,0 ° C a 40,0 ° C per 1 h in anticipo di riscaldare l'acqua.</li>
		<li>Modificare il blocco di paraffina acquisito da inclusione in paraffina; quindi posizionare il blocco di paraffina su morsetto di microtomo e bloccarlo. Nota: I bordi superiori ed inferiori del blocco paraffina dovrebbero essere modificati in orizzontale per evitare che il nastro di sezione da formando un arco e diventando in grado di fluire attraverso il canale di sezioni strette.</li>
		<li>Avvicinarsi rapidamente il campione di taglio di sezioni più spesse.</li>
		<li>Regolare ad uno spessore di sezione adeguata (10 µm per cervelli di topo adulto, reni di topo adulto e cervelli di topo di embrione, 4 µm per zebrafish occhi). Ruotare il volantino e le sezioni verranno immesso automaticamente il canale sezioni e bagno.</li>
		<li>Sezione il campione in una corsa di taglio lento e uniforme (Figura 3A). Nota: Utile della prima sezione sarà modificato dopo il volantino è stato interrotto per qualche minuto. Le lame monouso devono essere sostituite quando le sezioni hanno graffi consecutivi.</li>
		<li>Guida le sezioni nella camera del bagno a galleggiare in una linea (Figura 3B, 3C). Nota: Se nella sezione head aderisce alla parete della camera, è possibile utilizzare una guida di pennello per tenerlo galleggianti.</li>
		<li>Rispettare le sezioni su vetrini da microscopio. Dopo le sezioni dispiegarsi pienamente nel bagno d'acqua, sezioni separate diverse con le pinzette. Poi li pick up dal bagno di acqua sui vetrini al microscopio (Figura 3D). Nota: Sezioni dovrebbero essere prelevati dalla parte posteriore del bagno d'acqua, come quelli del canale di sezioni e l'anteriore del bagno d'acqua non sarà ancora completamente spiegati.</li>
		<li>Asciugare i vetrini mettendoli in un forno di 42 ° C durante la notte. Le diapositive quindi possono essere conservate a temperatura ambiente a tempo indeterminato.</li>
	</ol>
</li>
	<li>Utilizzare la colorazione ematossilina-eosina (HE) per motivi di valutazione del metodo migliorato8.</li>
</ol>

<ol>
	<li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li><li>Fujita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuron+differentiation+in+the+central+nervous+system+by+tritiated+thymidine+autoradiography.">Analysis of neuron differentiation in the central nervous system by tritiated thymidine autoradiography.</a> Journal of Comparative Neurology. 122 (3), 311-327 (1964).</li><li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+anti-roll+plate+as+a+remedy+for+the+ill-effects+of+electrical+charge+during+cryosectioning.">A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning.</a> Journal of Histochemistry and Cytochemistry. 37 (7), 1157-1160 (1989).</li><li>Onozato, M. L., Hammond, S., Merren, M., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+completely+automated+tissue-sectioning+machine+for+paraffin+blocks.">Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.</a> Journal of Clinical Pathology. 66 (2), 151-154 (2013).</li><li>Sabaliauskas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+zebrafish+histology.">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li><li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid-Cut:+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples.">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754 (2016).</li><li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles.">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438 (2010).</li><li>Cornell, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+and+Thin+Sectioning+of+Microbial+Colony+Biofilms+for+Microscopic+Analysis.">Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.</a> Journal of Visualized Experiments. (133), e57196 (2018).</li><li>Whiteland, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+T-cell+subsets+and+other+leukocytes+in+paraffin-embedded+rat+and+mouse+tissues+with+monoclonal+antibodies.">Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 313-320 (1995).</li><li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification.">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (125), e55796 (2017).</li><li>Venegas-Pino, D. E., Banko, N., Khan, M. I., Shi, Y., Werstuck, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+and+Characterization+of+Atherosclerotic+Lesions+in+the+Murine+Aortic+Sinus.">Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus.</a> Journal of Visualized Experiments. (82), e50933 (2013).</li><li>Lau, S. K., Chu, P. G., Weiss, L. 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Gli autori hanno un brevetto su questo dispositivo e non dichiarano interessi concorrenti.

Per migliorare la paraffina sezione morfologia e risolvere il problema del tempo sprecato durante il sezionamento di paraffina convenzionale, abbiamo creato una paraffina migliore metodo che combina taglio e dispiegarsi di sezionamento. Questo metodo migliorato si basa su semplici attrezzature che comprende un canale di sezione, un bagno di acqua e un riscaldatore con un interruttore di rilevazione di temperatura. La barra multifunzione sezione entra nel bagno d'acqua attraverso il canale di sezione e si svolge automatic...

Lo studio morfologico è importante nella ricerca biologica. Anche se la nuova tecnologia ha permesso ai ricercatori di osservare loro obiettivi direttamente dall'intero tessuto o organismi1,2,3, l'esemplare di taglio in sezioni sottili, seguirono dalla macchiatura, rimane il primario morfologia del metodo solo tessuto, ma anche proteine targeting direttamente nel tessuto. La microscopia chiara utilizza tre tipi di sezione: paraffina, congelato e semifini. Sebbene cryosectioning è comune per proteggere l'antigenicità del tessuto e la preparazione del campione è semplice, la morfologia del tessuto mantenuto è scadente e non adatto per sottile taglio4,5. Sezionamento di paraffina è il metodo più frequentemente utilizzato per l'esposizione di morfologia ben conservato. Come gli esemplari sono completamente disidratati e incorporati in cera, i blocchi di paraffina possono essere conservati a tempo indeterminato. Inoltre, paraffina sezionamento produce sezioni sottili che migliorare l'accesso della sonda biologico in ulteriori esperimenti e riducono la sovrapposizione di strati di cella nella direzione Z.
Tuttavia, paraffina convenzionale di sezionamento è noioso e richiede abilità dell'operatore. Sezioni della paraffina subiscono fissazione, disidratazione, incastonatura, taglio e galleggianti. È importante trasferire nastri sezione dal portalama a bagno d'acqua è necessaria, ma difficile per operatori junior. Specialmente in aria asciutta, i nastri di sezione torcerà elettricità statica e sono difficili da spiegare sulla superficie dell'acqua calda. Per migliorare la sezione qualità, inumidire la superficie di tessuto esposto tra microtomo lama passate, i blocchi di cera di raffreddamento immergendole in acqua con ghiaccio, o alzando l'umidità con un umidificatore vicino il microtomo sono consigliate6,7 . I più nuovi metodi per migliorare la paraffina sezionamento includono ibrido inclusione in paraffina, cryosectioning8e sezione commerciale trasferimento sistema assistenza9. Sebbene questi metodi migliorano parzialmente paraffina sezionamento velocità e qualità, fanno di sezionamento molto più ingombrante, e sistemi di trasferimento di sezione commerciale sono costosi.
In questo protocollo, dimostriamo come creare attrezzature passo dopo passo, semplice, economico e flessibile che possono essere collegata per il supporto della lama di un microtomo rotativo. Questa apparecchiatura è composta da un canale di sezione, un bagno di acqua e un riscaldatore con un interruttore di rilevazione di temperatura. Dopo il taglio, decine di sezioni fluiscono nella scanalatura della sezione e inserire il bagnomaria direttamente, così sta svolgendo automaticamente. Questo migliora l'efficienza di taglio di paraffina e rende questa tecnologia più conveniente. Utilizzando questo metodo, più adulti sezioni di hippocampal di topo, sezioni di rene di topo adulto, embrionale 15,5 sezioni giorno-vecchio (E15.5) del mouse del cervello e occhio di zebrafish adulto sezioni sono state raccolte in meno tempo e morfologicamente più intatto. Questo metodo può essere utilizzato anche per altri campioni di tessuto che richiedono paraffina accelerata di sezionamento, evitando perdita di distinzione di sezione.

<table><tbody><tr><td>Incubator</td><td>Boekel Scientific</td><td>133000-2</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>64-17-5</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>1330-20-7</td><td></td></tr><tr><td>Paraplast</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Heated Paraffin Embedding Module</td><td>Leica</td><td></td><td></td></tr><tr><td>Commercial acrylic board</td><td></td><td></td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>67-66-3</td><td></td></tr><tr><td>Tubular electric heating element(12 V 200 W)</td><td></td><td></td><td></td></tr><tr><td>Temperature controller(12 V 120 W)</td><td>Mingsuo</td><td>XH-W3002</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Neutral silicone sealant</td><td></td><td></td><td>Link the water channel with the microtome knife holder</td></tr><tr><td>Voltage transformer</td><td>Dearll</td><td>S-250-12</td><td></td></tr><tr><td>Disposable blade</td><td>Accu-Edge</td><td>4689</td><td></td></tr><tr><td>Hematoxylin</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Microslide</td><td>Sail Brand</td><td>7105</td><td></td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>10004160</td><td></td></tr><tr><td>Coverslip&amp;nbsp;</td><td>Citoglas</td><td>10212424C</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Xilong Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Water bath for paraffin sections</td><td>Leica</td><td></td><td></td></tr><tr><td>HistoCore Arcadia C&amp;nbsp;- Cold Plate</td><td>Leica</td><td></td><td></td></tr><tr><td>paraffin repellent spray&amp;nbsp;</td><td>Thermo Scientific</td><td>9990420</td><td></td></tr></tbody></table>

the cutting and floating method for paraffin-embedded tissue for sectioning

Sezionamento del tessuto paraffina-incastonato è ampiamente usato in istologia e patologia. Tuttavia, è noioso. Per migliorare questo metodo, diverse aziende commerciali hanno messo a punto sistemi di trasferimento di sezione complessa utilizzando acqua liquida. Per semplificare questa tecnologia, abbiamo creato un semplice metodo utilizzando attrezzature fatte in casa che combina taglio e galleggianti all'interno di una semplice camera termostatica; Pertanto, le sezioni immettere automaticamente il bagno d'acqua sulla superficie dell'acqua. L'ippocampo da cervelli di topo adulto, reni di topo adulto, cervelli embrionali del mouse e dello zebrafish adulto sono stati tagliati utilizzando paraffina convenzionale di sezionamento e il metodo proposto per il confronto. Analisi statistica dimostra che il nostro metodo migliorato salvato tempo e sezioni più alte qualità di prodotto. Inoltre, paraffina sezionamento di un esemplare intero in breve tempo è facile per gli operatori junior.

Il metodo migliorato ha aumentato il numero delle sezioni di paraffina intatto. Abbiamo testato questo metodo nuovo su tessuto ippocampale topo adulto, reni di topo adulto, cervelli embrionali del mouse e dello zebrafish. Acqua è stato aggiunto al serbatoio, e la temperatura dell'acqua è stata mantenuta tra 38,0 ° C a 40,0 ° C. Dopo una preparazione in serie i campioni di tessuto, sono stati sezionati e rispetto alla divisione convenzionale. Il nuovo metodo di evitata perdita di sezio...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Il taglio e il metodo di galleggiamento per tessuto paraffina-incastonato per il sezionamento

Qui, presentiamo un protocollo per migliorare la paraffina sezionamento. Questo metodo combina taglio e mobile utilizzando una semplice camera termostatica per evitare il processo di trasferimento richiesto secondo il metodo convenzionale. Di conseguenza, l'efficienza e il numero di sezioni della paraffina intatti furono notevolmente migliorate.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

the-cutting-floating-method-for-paraffin-embedded-tissue-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.2K Views.  Nanchang University. Qui, presentiamo un protocollo per migliorare la paraffina sezionamento. Questo metodo combina taglio e mobile utilizzando una semplice camera termostatica per evitare il processo di trasferimento richiesto secondo il metodo convenzionale. Di conseguenza, l'efficienza e il numero di sezioni della paraffina intatti furono notevolmente migliorate.

Video: The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Sezionamento sottile fetta di preparativi per immunoistochimica

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Ricerca

Biologia

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Sezione delle ghiandole mammarie per tutti gli attacchi per identificazione delle lesioni

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Ricerca sul cancro

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Inclusione in paraffina e sezionamento sottile di biofilm di colonie microbiche per l'analisi al microscopio

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Immunologia e infezioni

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Preparazione del tessuto cerebrale Primate non umano per la pre-embedding immunoistochimica e microscopia elettronica

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Neuroscienze

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

Valutazioni e analisi di tumore fetta espianti come prerequisito per la prova diagnostica

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Un metodo sezionamento migliorato per Recalcitrante tessuti vegetali Campioni

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Isole pancreatiche incorporamento per sezioni di paraffina

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Tecniche di microscopia per l'interpretazione della colonizzazione fungina nei tessuti delle piante micoeterotrofiche e della germinazione simbiotica dei semi

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

Formalin-Fixed Paraffin Embedded (FFPE) Tissue Preservation: A Method for Studying Tissue Morphology

Source: Nagaraj A.S., et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing.</a> J. Vis. Exp. (2018)
The following video describes how to preserve morphology of a tissue with formalin fixation and paraffin embedding.