The authors gratefully acknowledge their colleagues Jeffrey Tom and Aimin Song for peptide synthesis, Nianfeng Ge for TMA construction, Shari Lau for IHC staining, Melissa Edick for digital microscopic scanning, and Hai Ngu for digital image quantification.

1. Preparation of stock solution and tools
<ol>
	<li>Prepare 20 mL of a 25% w/v BSA solution by mixing 5 g BSA powder in 14 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Vortex as necessary to disperse the BSA powder.
	<ol>
		<li>Keep the solution overnight at 4 &#176;C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 25% w/v stock solution.</li>
	</ol>
	</li>
	<li>Prepare 20 mL of a 31.3% w/v BSA solution by mixing 6.26 g BSA powder in 13 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Keep the solution overnight at 4 &#176;C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 31.3% w/v stock solution.</li>
	<li>Preheat a heat block to 85 &#176;C. 
	NOTE: The protocol below creates peptide/BSA gels with volumes of 1.26-1.4 mL formed in 1.5 mL microcentrifuge tubes. To use smaller volumes, for example, when antigen stocks are limiting, prepare the gels in PCR tubes and use a thermocycler set to 85 &#176;C as a heat block.</li>
	<li>Test that the BSA/formaldehyde mixture forms a gel as expected by mixing 700 &#181;L of 25% BSA solution with 700 &#181;L of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles. 
	CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.</li>
	<li>Immediately after mixing the BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 &#176;C for 10 min. Remove the tube from the heat block and allow it to cool. Confirm that the gel has formed as expected.</li>
</ol>
2. Preparation and dilution of peptides
<ol>
	<li>Obtain 5-20 mg of lyophilized peptide of the desired sequence. 
	NOTE: The C-terminal 16 amino acids of the human ERBB2 intracellular domain recognized by 4B5 is TPTAENPEYLGLDVPV-COOH.
	<ol>
		<li>Add 4 amino acids to the N-terminus, acetyl-YGSG, and C-terminus, GSGC-amide to facilitate cross-linking of the peptide to BSA and provide spacing between the BSA molecule and the peptide epitope. 
		NOTE: The complete sequence is: acetyl-YGSGTPTAENPEYLGLDVPVGSGC-amide.</li>
		<li>If desired, use other N- and C-terminal amino acid sequences to extend the core peptide epitope. 
		NOTE: The impact of different sequences varies with different antibody/epitope combinations. The addition of the C-terminal peptide reduces the binding of some antibodies to C-terminal epitopes. In such cases, omit this sequence.</li>
		<li>Confirm that peptides from commercial sources are supplied at &#62;95% purity, the composition of which is confirmed by HPLC and mass spectrometry analysis.</li>
	</ol>
	</li>
	<li>Calculate the necessary volumes for the 5x (1.25 E-2 M) peptide stock solutions. Referring to Table 1, columns C-E, enter values for the antigen molecular weight (g/mole), percent antigen purity (0-100), and antigen mass (mg). 
	NOTE: The volume of solvent (in &#181;L) to resuspend the sample to achieve a stock solution of 1.25 E-2 M is 800 x antigen molecular weight x percent antigen purity/antigen mass.
	<ol>
		<li>Prepare and clearly label eight 1.5 mL microcentrifuge tubes. 
		NOTE: The tubes will contain 5x peptide stock in a solvent, 1x peptide stock in a solvent, five 10x serial dilutions of 2.5 E-4 M to 2.5 E-8 M peptide/BSA/formaldehyde gel, and a negative control gel containing BSA/formaldehyde lacking added antigen. All gel samples look identical. When preparing multiple sets of peptide dilutions at one time, take care to label and identify all tubes and processing cassettes correctly. Use color-coded microcentrifuge tubes and processing cassettes where possible to minimize misidentification.</li>
	</ol>
	</li>
	<li>Prepare a 5x peptide stock solution at 1.25 E-2 M by resuspending the entire mass of lyophilized peptide (20 mg for the ERBB2 peptides) in 60 &#181;L of the appropriate solvent. 
	NOTE: In this example, dimethylformamide (DMF) was added directly to the vendor&#39;s container.
	<ol>
		<li>Inspect the solution to ensure that the peptide is completely dissolved. If necessary, add additional solvent and/or sonicate the sample until the peptide is completely dissolved, taking care not to exceed the volume calculated in Table 1 for the 5x peptide stock. 
		CAUTION: DMF is toxic; use with appropriate precaution. 
		NOTE: Depending on the amino acid sequence, and the corresponding hydrophobicity and charge, peptides may be soluble in DMF, dimethyl sulfoxide (DMSO), pure water, or dilute solutions of acetic acid, formic acid, or ammonium bicarbonate. Peptide characteristics may be calculated using a variety of online tools17. Some peptide vendors may suggest solvents appropriate for specific sequences.</li>
		<li>Add solvent as necessary to bring the volume of the 5x peptide stock to the final volume calculated in Table 1. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. Peptide stock solutions can be stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Referring to Table 2, Column C, prepare 150 &#181;L of 1x peptide stock solution (2.5 E-3 M) by diluting 30 &#181;L of the 5x peptide stock into 120 &#181;L of solvent (DMF this example). Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s.</li>
	<li>Referring to Table 2, Column D, prepare 700 &#181;L of 5 E-4 M peptide/BSA solution (Dilution 1) by diluting 140 &#181;L of 1x peptide stock into 560 &#181;L of 31.3% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. 
	NOTE: The final BSA concentration of this solution is 25% (w/v).</li>
	<li>Referring to Table 2, Columns E-H, prepare four successive 10x serial dilutions of the 5 E-4 M peptide/BSA stock by adding 70 &#181;L of peptide/BSA solution to 630 &#181;L of 25% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. 
	NOTE: After this step, there will be five 10-fold serial dilutions of peptide (5 E-4 M to 5 E-8 M) in 25% BSA/PBS, pH 7.2. The first four samples will contain 630 &#181;L. The last sample will contain 700 &#181;L.</li>
	<li>Referring to Table 2, Column I, prepare a negative control BSA sample containing 700 &#181;L of 25% BSA/PBS, pH 7.2 (Figure 1A).</li>
</ol>
3. Preparing BSA-peptide gels
<ol>
	<li>Confirm that the heat block or thermocycler is stable at 85 &#176;C.</li>
	<li>Refer to Table 3, Columns B-E. Working one sample at a time, add to the first 25% BSA/peptide sample (Dilution 1) 630 &#181;L of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles. 
	CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.
	<ol>
		<li>After mixing the peptide/BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 &#176;C for 10 min. 
		NOTE: Mix the BSA-peptide solution and formaldehyde thoroughly, but do not spend more than 10 s pipetting the mixture before placing the sample on heat. Since formaldehyde cross-linking begins immediately, the gel may form differently if the procedure is varied for different samples. The final BSA concentration in these gels is 12.5% (w/v). Final BSA concentrations less than 10% may yield gels that do not solidify; final BSA concentrations greater than 16% may produce gels more brittle and difficult to section after processing.</li>
		<li>Repeat steps 3.2 and 3.2.1 for each of the dilutions 2-4.</li>
		<li>Repeat steps 3.2 and 3.2.1 for dilution 5, but add 700 &#181;L of 37% formaldehyde, a volume equal to the 700 &#181;L of BSA-antigen solution.</li>
		<li>Refer to Table 3 column Column G; repeat steps 3.2 and 3.2.1 for the negative control sample, adding 700 &#181;L of 37% formaldehyde, a volume equal to the 700 &#181;L of negative control BSA solution.</li>
	</ol>
	</li>
	<li>Remove the tubes from the heat block after 10-12 min. The heating time should be as consistent as possible for each sample. Allow the gels to cool on the benchtop for 5-10 min (Figure 1B).</li>
	<li>Using a clean, flexible disposable laboratory spatula, remove the gel sample in one piece from the microcentrifuge tube, and place it in a sealed container containing at least 15 mL of neutral buffered formalin (NBF), using a separate container of NBF for each sample.
	<ol>
		<li>Alternatively, cut off the bottom of the microcentrifuge tube with a new single edge razor blade, and push the gel out from the bottom with air or a suitable probe (Figure 1C-G). 
		&#8203;NOTE: The solidified formaldehyde/BSA gels can remain in the microcentrifuge tubes at room temperature for up to 24 h. Leaving the gels in the microcentrifuge tube for more than 24 h can cause them to become brittle and more difficult to process and section.</li>
	</ol>
	</li>
</ol>
4. Trimming, processing, and embedding BSA gels
<ol>
	<li>Trim the gel cone into cylindrical discs approximately 5 mm thick using a clean single edge razor (Figure 1H,I). Wrap the discs in a biopsy wrap, placing one larger gel disc into one cassette (to be used in the pilot study in step 5), and the remaining gel discs together into a second cassette (Figure 2A,E) for use in tissue microarray (TMA) construction in step 6. Place the wrapped gel discs in clearly labeled tissue processing cassettes.
	<ol>
		<li>Place the cassetted gels in at least 15 mL of 10% NBF per gel sample before processing, using a separate container of NBF for each sample. Gels can remain in 10% NBF for 6-48 h.</li>
	</ol>
	</li>
	<li>Process the gels in an automated histology tissue processor, following a large tissue schedule with pressure and vacuum. Each step takes 1 h: 10% NBF, 70% ethanol, 95% ethanol (repeat two times), 100% ethanol (repeat two times), xylenes (repeat three times), paraffin at 60 &#176;C (repeat three times). 
	NOTE: For investigators choosing to process samples manually, follow the same sequence of reagents and times.</li>
	<li>When the sample processing is completed, remove the cassettes from the tissue processor and move them to the paraffin embedding center.</li>
	<li>Unwrap gels from the biopsy wrap and embed the gels in paraffin. For each sample, embed one disk of gel in a small 15 mm x 15 mm mold (Figure 2B-D), and the remaining gel discs together in a second larger mold (Figure 2F-H). The first block with one sample will be used to test the peptide gel in a pilot study. The second block can be used for TMA construction.</li>
</ol>
5. Pilot evaluation of the peptide dilution series
<ol>
	<li>For each peptide dilution series, plan to create two glass slides containing a total of 6 separate sections: one section from each of the five dilution series samples, plus one section from the BSA-only negative control sample.
	<ol>
		<li>Onto the first glass slide, cut one 4 &#181;m thick section from each of the smaller blocks containing one gel disc with the three highest peptide concentrations (2.5 E-4 M to 2.5 E-6 M).</li>
		<li>Onto a second slide, cut one 4 &#181;m thick section from each block with the two lowest peptide concentrations (2.5 E-7 M and 2.5 E-8 M) and one section from the BSA-only control block. Record the order of the samples on the slides. 
		NOTE: Expect the paraffin-embedded gels to cut smoothly, producing uniform sections without fragmentation, tearing, or chattering artifact. If particular paraffin-embedded gel samples are difficult to section, briefly soak the block face in ice-cold distilled water before sectioning. If necessary, experiment with different soaking times or with different solutions (e.g., ammonia water).</li>
		<li>After sectioning, dry the slides at room temperature (about 23 &#176;C) for 24 h followed by 60 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Stain the two slides prepared for each peptide with the desired antibody according to standard IHC protocols. 
	NOTE: The primary antibody on-slide concentration used for rabbit monoclonal 4B5 in this demonstration was 1.5 ug/mL.
	<ol>
		<li>Expect to see a relatively uniform signal within each gel section, with the different gel samples showing a range of signal intensity corresponding to the peptide dilutions.</li>
	</ol>
	</li>
	<li>If the results for the pilot study are satisfactory, construct a TMA from the gel donor blocks containing different concentrations of peptide antigen, as described in the next steps of the protocol.</li>
</ol>
6. BSA gel TMA construction
<ol>
	<li>Construct a tissue microarray containing duplicate 1 mm diameter cores from donor blocks containing BSA gel alone and BSA gels containing all five dilutions of ERBB2 peptide. 
	NOTE: If desired, include BSA gels containing the same five dilutions of a non-target peptide as additional negative controls. If desired, include cores of representative ERBB2-expressing cell lines as positive controls.</li>
	<li>Cut 4 &#181;m thick sections of the TMA and stain with 1.5 ug/mL of anti-ERBB2/HER2/neu rabbit monoclonal 4B5 (see Table of Materials) according to laboratory-standard protocols.</li>
	<li>Assess the resulting stain intensity qualitatively by inspection or quantitatively by digital image scanning and analysis (Figure 3A,B). 
	NOTE: As digital image analysis is not the focus of this protocol, these steps are left for the reader to perform according to their preference.</li>
</ol>

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Charles A. Havnar, Kathy J. H&#246;tzel, Charles A. Jones, Carmina M. Espiritu, Linda K. Rangell, and Franklin V. Peale are employees and stockholders of Genentech and Roche. Their affiliates produce reagents and instruments used in this study.

This method allows the user to create uniform samples of known composition and antigen concentration as standards in IHC reactions, using materials and techniques familiar to most histology laboratories. The most crucial step is to identify the epitope to which the antibody of interest binds. This protocol describes using a linear peptide antigen from the ERBB2/HER2 ICD. The same protocol can be used to form BSA gels containing oligonucleotides, fluorescent labels, protein domains, or full-length proteins. This latter ap...

Immunohistochemistry (IHC) allows the sensitive and specific, spatially precise detection of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, IHC staining results may be affected by multiple variables, including warm and cold ischemia time, tissue fixation, tissue pretreatment, antibody reactivity and concentration, assay detection chemistry, and reaction times1. Accordingly, reproducible performance and interpretation of IHC reactions require rigorous control of these variables and the use of well-characterized positive and negative control samples. Frequently used controls include paraffin-embedded tissues or cultured cell lines known from independent analyses to express the antigen of interest, but such samples are not always available1. Furthermore, the expression levels of the target antigens in tissues and cell line controls are generally understood only qualitatively and may be variable. Controls containing reproducible, precisely known concentrations of target antigen can assist in the optimization of IHC reaction conditions. A general method, adaptable to a variety of antigen types in a physiologically relevant range of concentrations to create synthetic IHC control samples, has been described by the authors2. A detailed protocol is provided here for the creation and use of this type of standard.
Appropriate controls are essential for the valid interpretation of IHC assays1,3,4. Tissues, cultured cells, and peptide-coated substrates have been employed as IHC controls according to the investigators&#39; specific needs. The advantages and limitations inherent in using tissues as IHC controls have been extensively discussed1,4. For many antibodies, appropriate controls can be chosen from selected normal tissues containing cell populations expressing the target antigen over a wide dynamic range. Tissue controls are less suitable when the target antigen is not well-characterized concerning expression site or abundance, or when potentially cross-reacting antigens are co-expressed in the same cells or tissue sites. In these contexts, blocks of cultured cell lines expressing the antigen of interest can be helpful. For providing further evidence of target specificity, cell lines can be engineered to over-or under-express target antigens. For example, such an approach was recently used to evaluate a variety of anti-PD-L1 assays using a tissue microarray of isogenic cell lines expressing a range of PD-L1 antigen5. Practical limitations to the routine use of cell line blocks include the cost and time needed to produce sufficient cell numbers and the fact that the expression of some antigens may not be reliably consistent, even within clonal cell lines6. Synthetic peptides are a third option for IHC controls for antibodies that recognize short linear epitopes. Steven Bogen and colleagues have published extensively on the use of peptides coupled to the surface of glass slides7,8 and glass beads9. One study by this group demonstrated that quantitative analysis of peptide-based IHC controls could detect staining process variation missed by qualitative evaluation of tissue controls analyzed in parallel10. While standards using bead-based antigens could be widely applicable, many details are proprietary to the authors, limiting widespread adoption.
Another approach to IHC standards incorporates target antigens into artificially created protein gels. This concept was first demonstrated by Per Brandtzaeg in 1972 in a study in which normal rabbit serum was polymerized using glutaraldehyde11. Small blocks of the resulting gel were then soaked for 1-4 weeks in solutions containing the immunoglobulin antigens of interest at various concentrations. After alcohol fixation and paraffin embedding, sections of the resulting controls were shown to stain with intensities corresponding to the logarithm of the antigen solutions in which they had been soaked. Later, investigators prepared glutaraldehyde conjugates of specific amino acids in dilute BSA or brain homogenate solutions as positive controls in immune-electron microscopy studies12,13. More recent work investigated the use of gels made from formaldehyde-fixed protein solutions as surrogates for FFPE tissue in mass spectrometry analysis14. Another recent work investigated the structure and properties of gels formed by heating human or bovine serum albumin solutions at various concentrations and pH15. These authors found that serum albumin forms three types of gels differing in mechanical elasticity, secondary structure preservation, and fatty acid-binding capability depending on the experimental conditions. Together, these papers demonstrate the general feasibility of the approach employed here. Protein solutions of defined composition create tissue-like gels that can be further processed, sectioned, and stained using routine histological methods.
This protocol describes the formation of a synthetic IHC control made from bovine serum albumin (BSA) polymerized with heat and formaldehyde. The gels can incorporate a wide variety of antigens, including full-length proteins, protein domains, and linear peptides, as well as non-protein antigens&#160;including oligonucleotides2. This demonstration uses an example antigen&#160;a linear peptide encoding the C-terminal 16 amino acids of the human ERBB2 (HER2/neu) protein TPTAENPEYLGLDVPV-COOH (see Table of Materials). This sequence includes the epitopes recognized by three commercially available, diagnostically relevant antibodies&#160;including the Herceptest polyclonal reagent (ENPEYLGLDVP) and the monoclonal antibodies CB11 (AENPEYL) and 4B5 (TAENPEYLGL) (see Table of Materials)16.
The method demonstrated here employs readily available reagents using processes and techniques familiar to any practicing histology laboratory. The most significant limitation is the need to identify and purchase the target antigens, which can be accomplished in many cases at a relatively modest cost. Because these synthetic controls are of wholly defined composition and made with simple methods, they can be implemented in many laboratories with reproducible results. Their use may facilitate the objective, quantifiable evaluation of IHC staining results and allow greater intra- and inter-laboratory reproducibility.

<table><tbody><tr><td>Anti-HER2/neu clone 4B5</td><td>Ventana</td><td>5278368001</td><td></td></tr><tr><td>Biopsy Wraps</td><td>Leica</td><td>3801090</td><td></td></tr><tr><td>Bovine Serum Albumin, ultra pure</td><td>Cell Signaling Technology</td><td>BSA #9998</td><td></td></tr><tr><td>50 mL Conical Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Disposable base mold (15 mm x 15 mm)</td><td>Fisher</td><td>22-363-553</td><td></td></tr><tr><td>Disposable base mold&lt;br/&gt; (24 mm x 24 mm)</td><td>Fisher</td><td>22-363-554</td><td></td></tr><tr><td>Disposable spatula</td><td>VWR</td><td>80081-188</td><td></td></tr><tr><td>Eppendorf Thermomixer</td><td>Eppendorf</td><td>22331</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Electron Microscopy Sciences</td><td>15686</td><td></td></tr><tr><td>ERBB2 / HER2 peptide</td><td></td><td></td><td>UniProt P04626-1; a.a. 1240-55</td></tr><tr><td>Leica Autostainer XL</td><td>Leica</td><td>ST5010</td><td></td></tr><tr><td>Magnetic Stir Bar</td><td></td><td></td><td></td></tr><tr><td>NanoZoomer 2.0 HT whole slide imager</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>16004-128</td><td></td></tr><tr><td>Nuclease-free microfuge tubes 1.5 mL</td><td></td><td></td><td></td></tr><tr><td>Paraplast paraffin</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Peptide parameter calculator</td><td>Pep-Calc&lt;sup&gt;17&lt;/sup&gt;</td><td>https://www.pep-calc.com/</td><td></td></tr><tr><td>Peptide suppliers</td><td>ABclonal Science</td><td></td><td>Users should contact peptide vendors for details of mass, purity and cost.</td></tr><tr><td></td><td>Anaspec Peptide</td><td></td><td>Users should contact peptide vendors for details of mass, purity and cost.</td></tr><tr><td></td><td>CPC Scientific</td><td></td><td>Users should contact peptide vendors for details of mass, purity and cost.</td></tr><tr><td></td><td>New England Peptide</td><td></td><td>Users should contact peptide vendors for details of mass, purity and cost.</td></tr><tr><td>Phosphate Buffered Saline pH 7.2</td><td></td><td></td><td></td></tr><tr><td>Reagent Alcohol</td><td>Thermo Scientific</td><td>9111</td><td></td></tr><tr><td>Single Edge Razor</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Superfrost Plus positively charged microscope slides</td><td>Thermo Scientific</td><td>6776214</td><td></td></tr><tr><td>TMA Tissue Grand Master</td><td>3DHISTECH</td><td></td><td></td></tr><tr><td>Xylenes</td><td>VWR</td><td>89370-088</td><td></td></tr></tbody></table>

synthetic antigen controls for immunohistochemistry

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 &#176;C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.
This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Peptides should dissolve entirely in an appropriate solvent at room temperature to form an optically clear solution. If visible particulate material is still present after 30-60 min, it may be helpful to add additional volumes of the original solvent or an alternative solvent not exceeding the intended volume of the 5x peptide stock solution calculated in Table 1. Likewise, the combined peptide/BSA solution should remain translucent (Figure 1A).
P...

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Synthetic Antigen Controls for Immunohistochemistry

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires ...

synthetic-antigen-controls-for-immunohistochemistry

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

2.4K Views.  Genentech, Inc.. This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Video: Synthetic Antigen Controls for Immunohistochemistry

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Biology

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Neuroscience

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Cancer Research

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling ...

Developmental Biology

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Immunology and Infection

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Synthetic Antigen Controls for Immunohistochemistry

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Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Multiplex Cyclic Fluorescent Immunohistochemistry

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland