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 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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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

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production 

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

A Comprehensive Step-By-Step Guide for Exclusive Endoscopic Ossiculoplasty

Techniques of Endoscopic Ossiculoplasty

The utilization of endoscopes in modern otology has evolved from diagnostic purposes to the development of exclusive endoscopic ear surgery. This technique offers a panoramic view of the middle ear and provides an optimal magnification of the oval window region, the stapes' suprastructure, and the footplate, allowing great precision in prosthesis positioning during ossiculoplasty (OPL). Various techniques for ossicular chain reconstruction have been described in the literature. Either autologous or synthetic materials can be used for reconstruction. The use of a patient's own tissue minimizes the risk of implant rejection or extrusion of the prosthesis through the tympanic membrane. On the other hand, synthetic materials like titanium are light and rigid and do not require time-consuming prosthesis remodeling. The main objective of this article is to present a comprehensive step-by-step guide that serves as a surgical manual for exclusive endoscopic OPL. This guide will explain various forms of OPL using synthetic and autologous materials. The goal is to provide a comprehensive understanding of the various surgical techniques and support the integration into clinical practice.

Author Spotlight: Advancing Endoscopic Ossiculoplasty &#8211; Techniques, Innovations, and Practical Guidance for Clinical Integration

Exclusive Endoscopic ossiculoplasty (EEO) is a promising and minimally invasive approach for the treatment of conductive hearing loss due to ossicular chain disruptions and associated middle ear pathologies. Herein, step-by-step instructions and a discussion of various endoscopic ossiculoplasty techniques are presented.

The utilization of endoscopes in modern otology has evolved from diagnostic purposes to the development of exclusive endoscopic ear surgery. This ...

Exploring Uterine NK Cell Heterogeneity in the Endometrium with Multiplex IHC

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Author Spotlight: Multiplex Immunohistochemistry for Understanding Immune Regulation by Uterine NK Cells in Pregnancy

This study presents a pioneering method for quantifying uterine natural killer cell subsets during the window of implantation using advanced multiplexed fluorescent immunohistochemical staining techniques.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying ...