Name: synthetic antigen controls for immunohistochemistry
Uploaded: 2021-08-23T15:00:00
Description: Watch this Scientific Journal Video about Synthetic Antigen Controls for Immunohistochemistry at JoVE.com

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 ...

<ol>
	<li>Torlakovic, E. E., 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=Standardization+of+positive+controls+in+diagnostic+immunohistochemistry:+recommendations+from+the+International+Ad+Hoc+Expert+Committee.">Standardization of positive controls in diagnostic immunohistochemistry: recommendations from the International Ad Hoc Expert Committee.</a> Applied Immunohistochemistry and Molecular Morphology. 23 (1), 1-18 (2015).</li><li>H&#246;tzel, K. J., 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=Synthetic+antigen+gels+as+practical+controls+for+standardized+and+quantitative+immunohistochemistry.">Synthetic antigen gels as practical controls for standardized and quantitative immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 67 (5), 309-334 (2019).</li><li>Hewitt, S. M., Baskin, D. G., Frevert, C. W., Stahl, W. L., Rosa-Molinar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunohistochemistry:+The+histochemical+society's+standards+of+practice+for+validation+of+immunohistochemical+assays.">Controls for immunohistochemistry: The histochemical society's standards of practice for validation of immunohistochemical assays.</a> Journal of Histochemistry and Cytochemistry. 62 (10), 693-697 (2014).</li><li>Torlakovic, E. E., 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=Standardization+of+negative+controls+in+diagnostic+immunohistochemistry:+recommendations+from+the+international+ad+hoc+expert+panel.">Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel.</a> Applied Immunohistochemistry and Molecular Morphology. 22 (4), 241-252 (2014).</li><li>Martinez-Morilla, S., 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=Quantitative+assessment+of+PD-L1+as+an+analyte+in+immunohistochemistry+diagnostic+assays+using+a+standardized+cell+line+tissue+microarray.">Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.</a> Laboratory Investigation. 100, 4-15 (2020).</li><li>Ben-David, U., 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=Genetic+and+transcriptional+evolution+alters+cancer+cell+line+drug+response.">Genetic and transcriptional evolution alters cancer cell line drug response.</a> Nature. 560 (7718), 325-330 (2018).</li><li>Sompuram, S. R., 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=Synthetic+peptides+identified+from+phage-displayed+combinatorial+libraries+as+immunodiagnostic+assay+surrogate+quality-control+targets.">Synthetic peptides identified from phage-displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets.</a> Clinical Chemistry. 48 (3), 410-420 (2002).</li><li>Sompuram, S. R., 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=A+novel+quality+control+slide+for+quantitative+immunohistochemistry+testing.">A novel quality control slide for quantitative immunohistochemistry testing.</a> Journal of Histochemistry and Cytochemistry. 50 (11), 1425-1433 (2002).</li><li>Sompuram, S. R., Vani, K., Tracey, B., Kamstock, D. A., Bogen, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+immunohistochemistry:+A+new+reference+control+for+detecting+staining+problems.">Standardizing immunohistochemistry: A new reference control for detecting staining problems.</a> Journal of Histochemistry and Cytochemistry. 63 (9), 681-690 (2015).</li><li>Vani, 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=Levey-Jennings+analysis+uncovers+unsuspected+causes+of+immunohistochemistry+stain+variability.">Levey-Jennings analysis uncovers unsuspected causes of immunohistochemistry stain variability.</a> Applied Immunohistochemistry and Molecular Morphology. 24 (10), 688-694 (2016).</li><li>Brandtzaeg, P. <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+immunofluorescence+with+artificial+sections+of+selected+antigenicity.">Evaluation of immunofluorescence with artificial sections of selected antigenicity.</a> Immunology. 22, 177-183 (1972).</li><li>Matute, C., Streit, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+demonstrating+GABA-Iike+immunoreactivity.">Monoclonal antibodies demonstrating GABA-Iike immunoreactivity.</a> Histochemistry. 86, 147-157 (1986).</li><li>Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembedding+light-+and+electron+microscopic+immunocytochemistry+of+amino+acids:+description+of+a+new+model+system+allowing+identical+conditions+for+specificity+testing+and+tissue+processing.">Postembedding light- and electron microscopic immunocytochemistry of amino acids: description of a new model system allowing identical conditions for specificity testing and tissue processing.</a> Experimental Brain Research. 69, 167-174 (1987).</li><li>Fowler, C. B., Cunningham, R. E., O'Leary, T. J., Mason, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Tissue+surrogates'+as+a+model+for+archival+formalin-fixed+paraffin-embedded+tissues.">'Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues.</a> Laboratory Investigation. 87, 836-846 (2007).</li><li>Arabi, S. H., 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=Serum+albumin+hydrogels+in+broad+pH+and+temperature+ranges:+characterization+of+their+self-assembled+structures+and+nanoscopic+and+macroscopic+properties.">Serum albumin hydrogels in broad pH and temperature ranges: characterization of their self-assembled structures and nanoscopic and macroscopic properties.</a> Biomaterials Science. 6 (3), 478-492 (2018).</li><li>Schrohl, A. -. S., Pedersen, H. C., Jensen, S. S., Nielsen, S. L., Br&#252;nner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+epidermal+growth+factor+receptor+2+(HER2)+immunoreactivity:+specificity+of+three+pharmacodiagnostic+antibodies.">Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies.</a> Histopathology. 59, 975-983 (2011).</li><li>Lear, S., Cobb, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pep-Calc.com:+a+set+of+web+utilities+for+the+calculation+of+peptide+and+peptoid+properties+and+automatic+mass+spectral+peak+assignment.">Pep-Calc.com: a set of web utilities for the calculation of peptide and peptoid properties and automatic mass spectral peak assignment.</a> Journal of Computer-Aided Molecular Design. 30, 271-277 (2016).</li><li>Camp, R. L., Chung, G. G., Rimm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+subcellular+localization+and+quantification+of+protein+expression+in+tissue+microarrays.">Automated subcellular localization and quantification of protein expression in tissue microarrays.</a> Nature Medicine. 8 (11), 1323-1327 (2002).</li><li>Angelo, 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=Multiplexed+ion+beam+imaging+of+human+breast+tumors.">Multiplexed ion beam imaging of human breast tumors.</a> Nature Medicine. 20 (4), 436-442 (2014).</li><li>Buus, S., 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-resolution+mapping+of+linear+antibody+epitopes+using+ultrahigh-density+peptide+microarrays.">High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays.</a> Molecular and Cellular Proteomics. 11 (12), 1790-1800 (2012).</li><li>Forsstr&#246;m, B., 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=Proteome-wide+epitope+mapping+of+antibodies+using+ultra-dense+peptide+arrays.">Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays.</a> Molecular and Cellular Proteomics. 13 (6), 1585-1597 (2014).</li><li>Forsstr&#246;m, B., 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=Dissecting+antibodies+with+regards+to+linear+and+conformational+epitopes.">Dissecting antibodies with regards to linear and conformational epitopes.</a> PLoS One. 10 (3), 0121673 (2015).</li><li>Prasad, B., 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=Toward+a+consensus+on+applying+quantitative+liquid+chromatography-tandem+mass+spectrometry+proteomics+in+translational+pharmacology+research:+A+white+paper.">Toward a consensus on applying quantitative liquid chromatography-tandem mass spectrometry proteomics in translational pharmacology research: A white paper.</a> Clinical Pharmacology and Therapeutics. 106 (3), 525-543 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			(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-Calc17</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...

Watch this Scientific Journal Video about Synthetic Antigen Controls for Immunohistochemistry at JoVE.com

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 ...

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