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

作者感谢他们的同事杰弗里·汤姆和艾明·宋的肽合成，格念峰的TMA构建，刘莎莉的IHC染色，梅丽莎·埃迪克的数字显微扫描，海鸥的数字图像定量。

1. 储备溶液和工具的制备
<ol><li>通过将5g BSA粉末与14 mL PBS混合，在50 mL锥形管中混合pH 7.2直至均匀分散，制备20 mL 25%w / v BSA溶液。根据需要涡旋以分散BSA粉末。<ol><li>将溶液保持在4°C过夜以使其完全溶解。用PBS将最终体积调节至20 mL，制成25%w / v储备溶液。</li>
	</ol></li>
	<li>通过将6.26g BSA粉末与13 mL PBS混合，在50 mL锥形管中混合pH 7.2，直到均匀分散，制备20 mL 31.3%w / v BSA溶液。将溶液保持在4°C过夜?...

<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. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>

该方法允许用户使用大多数组织学实验室熟悉的材料和技术，创建已知成分和抗原浓度的均匀样品作为IHC反应的标准。最关键的一步是鉴定目标抗体与之结合的表位。该协议描述了使用来自ERBB2 / HER2 ICD的线性肽抗原。相同的方案可用于形成含有寡核苷酸，荧光标记，蛋白质结构域或全长蛋白质的BSA凝胶。后一种方法有助于抗体与构象表位结合，这些表位不是由单个线性肽序列重建的。例如，含有...

免疫组化 （IHC） 允许在福尔马林固定的石蜡包埋 （FFPE） 组织切片中对靶抗原进行灵敏、特异性、空间精确的检测。然而，IHC染色结果可能受到多种变量的影响，包括冷热缺血时间、组织固定、组织预处理、抗体反应性和浓度、测定检测化学和反应时间1。因此，IHC反应的可重复性能和解释需要严格控制这些变量，并使用表征良好的阳性和阴性对照样品。常用的对照包括石蜡包埋组织或从独立分析中已知的培养细胞系来表达感兴趣的抗原，但此类样品并不总是可用的1。此外，靶抗原在组织和细胞系对照中的表达水平通常只能定性地理解，并且可能是可变的。含有可重复的、精确已知的目标抗原浓度的对照有助于优化IHC反应条件。作者2已经描述了一种通用方法，该方法适用于在生理相关浓度范围内的各种抗原类型以创建合成的IHC对照样品。这里提供了创建和使用此类标准的详细协议。
适当的对照对于IHC测定的有效解释至关重要1，3，4。根据研究人员的特定需求，组织，培养的细胞和肽包被的底物用作IHC对照。使用组织作为IHC对照组的固有优点和局限性已被广泛讨论1，4。对于许多抗体，可以从选定的正常组织中选择合适的对照，这些组织含有在宽动态范围内表达靶抗原的细胞群。当靶抗原在表达位点或丰度方面不能很好地表征时，或者当潜在的交叉反应抗原在相同的细胞或组织位点共表达时，组织对照不太合适。在这些情况下，表达感兴趣抗原的培养细胞系块可能会有所帮助。为了提供靶标特异性的进一步证据，可以将细胞系设计为过度表达或表达不足的靶标抗原。例如，最近使用这种方法来评估各种抗PD-L1测定，使用表达一系列PD-L1抗原5的等基因细胞系的组织微阵列。常规使用细胞系块的实际限制包括产生足够细胞数量所需的成本和时间，以及某些抗原的表达可能不可靠地一致，即使在克隆细胞系6中也是如此。合成肽是识别短线性表位的抗体的IHC对照的第三种选择。Steven Bogen及其同事已经发表了大量关于将肽耦合到载玻片7，8 和玻璃珠9表面的用途。该组的一项研究表明，基于肽的IHC对照的定量分析可以检测通过对平行分析的组织对照进行定性评估而遗漏的染色过程变异10。虽然使用基于磁珠的抗原的标准品可能广泛适用，但许多细节是作者专有的，限制了广泛采用。
IHC标准的另一种方法是将靶抗原掺入人造蛋白质凝胶中。1972年，Per Brandtzaeg在一项研究中首次证明了这一概念，其中使用戊二醛11聚合了正常的兔血清。然后将所得凝胶的小块在含有不同浓度的目标免疫球蛋白抗原的溶液中浸泡1-4周。在酒精固定和石蜡包埋之后，显示所得对照的部分染色强度对应于它们浸泡的抗原溶液的对数。后来，研究人员在稀释的BSA或脑匀浆溶液中制备了特定氨基酸的戊二醛偶联物，作为免疫电子显微镜研究中的阳性对照12，13。最近的工作研究了在质谱分析中使用由甲醛固定蛋白质溶液制成的凝胶作为FFPE组织的替代品14。最近的另一项工作研究了通过加热人或牛血清白蛋白溶液以各种浓度和pH15形成的凝胶的结构和性质。这些作者发现，血清白蛋白形成三种类型的凝胶，根据实验条件的不同，它们在机械弹性，二级结构保存和脂肪酸结合能力方面有所不同。这些论文共同证明了这里采用的方法的一般可行性。具有明确成分的蛋白质溶液产生组织样凝胶，可以使用常规组织学方法进一步处理，切片和染色。
该协议描述了由牛血清白蛋白（BSA）与热和甲醛聚合而成的合成IHC对照的形成。凝胶可以包含多种抗原，包括全长蛋白、蛋白结构域和线性肽，以及包括寡核苷酸2在内的非蛋白抗原。本演示使用了一个抗原，该肽是一种线性肽，编码人ERBB2（HER2 /neu）蛋白TPTAENPEGLDV-COOH的C端16个氨基酸（参见 材料表）。该序列包括由三种市售的诊断相关抗体识别的表位，包括赫西普斯特多克隆试剂（ENPEYLGLDVP）和单克隆抗体CB11（AENPEYL）和4B5（绦柏）（见 材料表）16。
这里演示的方法使用任何实践组织学实验室都熟悉的过程和技术来使用现成的试剂。最重要的限制是需要鉴定和购买靶抗原，这在许多情况下可以以相对适度的成本完成。由于这些合成对照具有完全定义的成分并用简单的方法制成，因此它们可以在许多实验室中实施，结果可重复。它们的使用可能有助于对IHC染色结果进行客观，可量化的评估，并允许更高的实验室内和实验室间重现性。

<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

免疫组化 （IHC） 检测为蛋白质表达模式提供了有价值的见解，需要对蛋白质表达模式进行可靠的解释，需要对照样品进行明确表征的阳性和阴性对照样品。由于并不总是可以使用适当的组织或细胞系对照，因此创建合成IHC对照的简单方法可能是有益的。此处描述了这种方法。它适用于各种抗原类型，包括蛋白质，肽或寡核苷酸，浓度范围广。该协议解释了创建合成抗原对照所需的步骤，以来自人成红细胞癌基因B2（ERBB2 / HER2）细胞内结构域（ICD）的肽为例，由各种诊断相关抗体识别。将牛血清白蛋白（BSA）溶液中HER2 ICD肽的连续稀释液与甲醛混合，并在85°C下加热10分钟以固化并交联肽/ BSA混合物。所得凝胶可以像组织一样进行处理、切片和染色，产生一系列已知抗原浓度的样品，涵盖广泛的染色强度。
这个简单的方案与常规组织学实验室程序一致。该方法仅要求用户具有足够数量的所需抗原。编码相关表位的重组蛋白、蛋白结构域或线性肽可以在局部或商业上合成。产生内部抗体的实验室可以保留免疫抗原的等分试样作为合成对照靶标。在各种浓度下创建明确定义的阳性对照的机会使用户能够评估实验室内和实验室间的分析性能，深入了解其测定的动态范围和线性度，并针对其特定的实验目标优化测定条件。

肽应在室温下完全溶解在适当的溶剂中以形成光学透明的溶液。如果可见颗粒材料在30-60分钟后仍然存在，则添加额外体积的原始溶剂或替代溶剂可能会有所帮助，不超过 表1中计算的5x肽储备溶液的预期体积。同样，组合的肽/ BSA溶液应保持半透明（图1A）。
肽/ BSA凝胶样品在用37%甲醛加热后应形成不透明的橡胶块（图1B-I</str...

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

Synthetic Antigen Controls for Immunohistochemistry

这项工作记录了一种为免疫组化创建合成抗原对照的简单方法。该技术适用于各种浓度的抗原。这些样品为评估测定内和测定间的性能和再现性提供了参考。

用于免疫组化的合成抗原对照

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

BSA抗原凝胶是表征良好的可重现IHC标准品，可以补充现有对照。该协议使用标准组织学和IHC试剂和方法来制备已知成分的可重现标准品。这些对照为不同的实验室提供了校准和标准化许多诊断相关检测的IHC检测的机会。找到合适的溶剂来溶解肽或蛋白质很重要。在不引入气泡的情况下快速混合抗原和甲醛溶液可能很棘手。首先，通过在50毫升锥形管中将5克BSA粉末与14毫升PBS混合，直到均匀分布，制备20毫升25%体积重量的BSA溶液。涡旋溶液以溶解BSA粉末。将溶液在四摄氏度下保持过夜以完全溶解。第二天，用PBS将最终体积调整为20毫升。同样，通过将6.26克BSA粉末与13毫升PBS混合，制备20毫升31.3%体积重量的BSA溶液。孵育过夜完全溶解后，用PBS将最终体积调节至20毫升。要测试BSA甲醛混合物是否形成凝胶，请将加热块预热至85摄氏度。然后将700微升25%BSA溶液与700微升37%甲醛混合。在 5 到 10 秒内上下移液 5 次，充分混合，不会产生气泡。混合后，立即将封闭的微量离心管放入预热至85摄氏度的加热块中。10分钟后，将管子从加热块上取下。让它冷却并确认凝胶已按预期形成。准备并清楚地标记八个 1.5 毫升微量离心管。然后制备5x肽储备溶液，通过将全部自由化肽重悬于60微升的适当溶剂中，加入12.5毫摩尔浓度。观察溶液以确保肽完全溶解。然后根据需要添加额外体积的溶剂，根据肽的分子量、质量和纯度制备 12.5 毫摩尔溶液。在本例中，5x肽储备液的最终体积为626.9微升。其他肽样品将需要不同的最终体积。接下来，准备 150 微升 1x 肽储备液，通过将 30 μL 的 5x 肽储备液稀释到 120 微升溶剂中来加入 2.5 毫摩尔浓度。将溶液涡旋五秒钟，并在室温下离心。接下来，通过将 140 微升 1x 肽储备液稀释到 560 微升 31.3% BSA 溶液中，制备 700 微升 0.5 毫摩尔肽 BSA 溶液。涡旋后，在室温下离心溶液。类似地，通过将70微升肽BSA溶液加入630微升25%BSA溶液中，制备四次连续10倍连续稀释的肽BSA储备液。要制备BSA肽凝胶，请以1比1的比例向肽BSA稀释液中加入37%甲醛，一次处理一个样品。在 5 到 10 秒内上下移液 5 次，充分混合，不会产生气泡。混合后，将封闭的微量离心管置于85摄氏度的加热块中10分钟。制备并加热所有肽BSA和甲醛溶液后，让凝胶在工作台上冷却5至10分钟。接下来，使用干净、灵活的一次性实验室刮刀，从微量离心管中取出凝胶样品。并将其放入装有至少15毫升中性缓冲福尔马林的密封容器中，每个样品使用单独的容器。或者，使用新的单刃剃须刀片，切下微量离心管的底部，然后用空气或合适的探针将凝胶从底部推出。使用干净的单刃剃须刀将凝胶锥修剪成约5毫米厚的圆柱形圆盘。将圆盘包裹在活检包装中后，将一个较大的凝胶盘放入一个凝胶盒中，将剩余的凝胶盘一起放入第二个凝胶盒中，用于组织微阵列构建。在处理之前，将盒式凝胶放入每个凝胶至少15毫升10%中性缓冲福尔马林中，每个样品使用单独的容器。接下来，在自动组织学组织处理器中按照压力和真空的大组织计划处理凝胶。样品处理完成后，从组织处理器中取出暗盒并将它们移动到石蜡包埋中心。从活检包装中解开凝胶后，将凝胶嵌入石蜡中，或者每个样品将一盘凝胶嵌入15×15毫米的小模具中。并将剩余的凝胶盘放在第二个较大的模具中。对于每个肽稀释系列，创建两个载玻片，总共包含六个独立的切片。五个稀释系列样品中每个样品的一个切片和一个仅BSA阴性对照样品的切片。切片并干燥载玻片后，用所需的一抗对为每种肽制备的两张载玻片进行染色。根据标准的免疫组织化学方案，期望在每个凝胶切片内看到相对均匀的信号。不同的凝胶样品显示出对应于肽稀释度的信号强度范围。对于BSA凝胶组织微阵列构建，切割组织微阵列的四个微米厚的切片，并根据实验室标准方案用兔单克隆抗体染色。通过检测定性评估产生的污渍强度，或定量购买数字图像扫描和分析。在与适当的抗体反应后，阴性对照BSA仅凝胶样品显示出最小的信号。单个凝胶样品中的信号强度相对均匀，并随着抗原浓度的增加而增加。超过 10% 的 MDA-MB-175 细胞显示微弱且完全圆周膜染色。相比之下，超过10%的SKBR-3细胞显示出强烈的完全圆周膜染色一旦加入甲醛，就会迅速起作用，因为即使在室温下溶液也会开始凝胶化。我们在测试新抗体时使用这种方法进行IHC对照，以使我们相信即使测试抗体未显示信号，检测也能按预期进行。组织或细胞系对照本质上是可变的。合成抗原对照使我们能够更精确地测量改变特定ISD反应条件的影响。

Research

JoVE Journal

Medicine

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

利用视觉和叙事方法，以实现公平的进程中，在临床护理

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

科研

医学

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

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

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

增强细胞凋亡和自噬的诱导合成一种新型的C-1 7-deoxypancratistatin的类似物在人类乳癌细胞和神经母细胞瘤与他莫昔芬

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

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

鉴定的使用神经影像学数据的与疾病-相关的的空间协方差模式

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

大鼠建立压力超负荷心肌肥厚模型在升主动脉缩窄

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

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

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

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

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

建模与嗅觉药物传送的模拟与鼻吸入气雾剂药品的被动和主动控制

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

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

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

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

植入遥测心电图和血压递质组合以确定意识小鼠的自发压力反射敏感性

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...