Immunohistochemistry was performed essentially as described in our previous report [17 (link)] with a mouse anti-Nestin monoclonal antibody diluted 1:200 (Millipore, Temecula, CA; catalog number: MAB353). The Envision + Horseradish Peroxidase System (Dako Japan, catalog number: K5027) was used along with a rabbit anti-human PGP9.5 antibody diluted to 1:400 (Ultra Clone Ltd., Isle of Wight, UK; catalog number: RA95101), a rat anti-mouse CD31 antibody diluted to 1:50 (BD Pharmingen, San Diego, CA; catalog number: 553,370), and a rat anti-Ki67 monoclonal antibody diluted to 1:100 for the cell proliferation assay (Dako Japan, Tokyo, Japan; catalog number: M7249). The avidin-biotin peroxidase complex (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA) formation was determined using biotinylated anti-rabbit or anti-rat immunoglobulin G (Vector Laboratories; catalog number: BA-4000).
Envision horseradish peroxidase system
Manufactured by Agilent Technologies
Sourced in Japan, United States, Denmark
The EnVision+ horseradish peroxidase system is a detection system used in immunoassays. It employs horseradish peroxidase enzyme conjugates to amplify the signal generated during the assay, enhancing the sensitivity of the detection process.
Automatically generated - may contain errors
Lab products found in correlation
Dako autostainer, by Agilent Technologies (5 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Aperio scanscope, by Leica (2 mentions)
Aperio imagescope version 11, by Leica (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Rat anti mouse cd31 antibody, by BD (1 mentions)
K4007, by Agilent Technologies (1 mentions)
Fitc conjugated anti rabbit igg, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Target retrieval solution ph 9, by Agilent Technologies (1 mentions)
Aperio cs2 slide scanner, by Leica (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Envision system hrp labelled polymer anti mouse, by Agilent Technologies (1 mentions)
Biotinylated anti rat igg, by Vector Laboratories (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Ix73 inverted microscope, by Olympus (1 mentions)
Anti cd24, by Thermo Fisher Scientific (1 mentions)
Anti ck5 6, by Agilent Technologies (1 mentions)
Dab substrate chromogen system, by Agilent Technologies (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
16 protocols using envision horseradish peroxidase system
1
Immunohistochemical Characterization of Cellular Markers
2
Immunohistochemical PRAME Staining Protocol
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EOC TMA were described previously [42 (link), 56 (link)]. TMA sections were cut to 4 μm, placed on charged slides, and dried at 60°C for one hour. Slides were cooled to room temperature, de-paraffinized in three changes of xylene, and rehydrated using graded alcohols. For antigen retrieval, slides were heated in a steamer for 40 minutes in EDTA buffer, pH = 8, (Lab Vision), followed by a 20-minute cool down. Endogenous peroxidase was quenched with aqueous 0.3% H2O2 for 10 minutes and washed with PBS/T. Slides were loaded on a DAKO autostainer (Dako) and serum-free protein block was applied for 5 minutes, blown off, and then PRAME primary antibody (Sigma, catalog no. HPA045153) was applied at 1.3 μg/ml for one hour. A matched isotype antibody was applied to a replicate slide, in place of primary antibody, as a negative control. The EnVision+ horseradish peroxidase system (Dako) and DAB chromogen were used for visualization. Slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped.
3
Automated Digital Pathology Analysis of Immune Markers
4
Automated IHC and Digital Pathology Analysis
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IHC assays and automated digital pathology analysis were performed on formalin-fixed paraffin-embedded tissue blocks. Tissue sections (4–5 μm) were prepared on slides loaded on a DAKO autostainer (Dako) and after serum-free protein block the respective primary antibodies for CD3, CD4, CD8, Foxp3, IDO1, PD-1, and PD-L1 were applied separately. The EnVision+ horseradish peroxidase system (Dako) and DAB (3,3-diaminobenzidine) chromogen were used for visualization. Slides were digitally scanned using Aperio Scanscope (Aperio Technologies, Inc.) with 20 × bright-field microscopy. These images were then accessible using Spectrum (Aperio Technologies, Inc.), a web-based digital pathology information management system. Slide images are automatically associated to a digital slide created in the Digital Slide table in Aperio eSlide Manager. Once slides are scanned, Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc.) was used to view images for image analysis. An outline of the tumor and the size of the analysis area were defined, and the lymphocytes were counted using an optimized algorithm for each stain and results were normalized to number of lymphocytes per square millimeter.
5
Quantification of Proliferating Hepatocytes
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Formalin-fixed paraffin-embedded sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, and demasked in a microwave oven for 24 min in Tris/EDTA (TE) buffer (pH = 9.1). Antibodies for immunohistochemistry were Rabbit monoclonal anti-Ki67 (1:500, SP6, GTX16667, Cytotech ApS, Hellebæk, Denmark). Sections were counterstained with hematoxylin, and the antigen-antibody reaction was visualized with Dako EnVision horse radish peroxidase system using 3,3′-diaminobenzidine as the chromogen (K4007, DAKO North America Inc., Camarillo, CA, USA).
Livers from four individual animals of each genotype and age group were used for quantification of proliferating hepatocytes. From each animal, two representative liver tissue blocks were used for obtaining four randomly collected slices including at least 1000 hepatocytes from each liver (imaged with ×40 obj.). The total number of proliferative hepatocytes, given as the sum of all positively Ki67 labeled hepatocyte nuclei, and the semi-quantitative estimation of the average number of hepatocyte nuclei included in a magnification field were determined by counting.
Livers from four individual animals of each genotype and age group were used for quantification of proliferating hepatocytes. From each animal, two representative liver tissue blocks were used for obtaining four randomly collected slices including at least 1000 hepatocytes from each liver (imaged with ×40 obj.). The total number of proliferative hepatocytes, given as the sum of all positively Ki67 labeled hepatocyte nuclei, and the semi-quantitative estimation of the average number of hepatocyte nuclei included in a magnification field were determined by counting.
6
Immunohistochemical and Immunofluorescence Analysis of TIM-1 and TIM-4 in Hepatocellular Carcinoma
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The tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich) and then cut into 4-um sections, which were processed for immunohistochemistry as previously described [28 (link)]. Then, the sections were incubated with a primary antibody against human TIM-1 (Sigma-Aldrich). The staining was detected by a Dako-Cytomation Envision Horseradish Peroxidase System (DakoCytomation). Two independent observers who were blinded to the clinical outcomes evaluated the immunohistochemical variables.
For the immunofluorescence analysis, paraffin-embedded human HCC sections were stained with mouse anti-CD20 (Abcam) plus rabbit anti TIM-4 (Abcam) antibodies, followed by FITC-conjugated anti-rabbit IgG plus CY3-conjugated anti-mouse IgG antibodies (Abcam). Positive cells were detected by confocal microscopy (LSM 510, Carl Zeiss; ZEN 2010 software). All antibodies are listed in Additional file4 : Table S4.
For the immunofluorescence analysis, paraffin-embedded human HCC sections were stained with mouse anti-CD20 (Abcam) plus rabbit anti TIM-4 (Abcam) antibodies, followed by FITC-conjugated anti-rabbit IgG plus CY3-conjugated anti-mouse IgG antibodies (Abcam). Positive cells were detected by confocal microscopy (LSM 510, Carl Zeiss; ZEN 2010 software). All antibodies are listed in Additional file
7
Automated IHC Profiling of T-Cell Subsets
8
Immunohistochemical Analysis of DNA Damage Response
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The study was carried out in accordance with City of Hope's and Vanderbilt University's policies governing the use of patient specimens and PDX tissues, respectively. 5-μm-thick serial sections were made from formalin-fixed, paraffin-embedded patient tumors or PDX tumors. The γH2A.X-specific antibody was purchased from Cell Signaling Technology. Antibodies specific to Chk1 and phosphorylated Rad17 were purchased from LifeSpan Biosciences. The staining was detected by the EnVision + horseradish peroxidase system purchased from Dako (Carpinteria, CA). Counterstaining was with 50% Mayer's hematoxylin (DAKO) for 3 min. Slides were visualized and images were acquired on an Aperio® CS2 slide scanner under a 20 × objective lens (Leica, Buffalo Grove, IL).
9
Laryngeal Carcinoma Tissue Microarray Analysis
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The tissue microarrays (LP804) were purchased from U.S. Biomax, Inc. to detect POSTN, S100A2, KRT17, and FN1 in cores from 60 laryngeal carcinoma tissues and 20 non-tumor tissues. Graded ethanol was used to deparaffinize the cores. H2O2 was used to quench the endogenous peroxidase activity. After blocking, antibodies against S100A2 (1: 90), KRT17 (1: 200), POSTN (1:150), and FN1 (1: 200) were added and incubated at 4°C overnight. Detection was performed with the Envision/horseradish peroxidase system (Dako-Cytomation, Denmark) [13 (link)].
Semi-quantification of protein expression was defined by scoring criteria as previously described [14 (link)]. The positive cells (%) and staining intensity (scale 0–3) were checked, which were then multiplied to yield a score from 0 to 300. In order to maintain consistency, the same qualified pathologist gave interpretations for all IHC data.
Semi-quantification of protein expression was defined by scoring criteria as previously described [14 (link)]. The positive cells (%) and staining intensity (scale 0–3) were checked, which were then multiplied to yield a score from 0 to 300. In order to maintain consistency, the same qualified pathologist gave interpretations for all IHC data.
10
Immunohistochemical Staining Protocol
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IHC staining was performed using EnVision+/Horseradish Peroxidase system (DAKO). Formalin-fixed, paraffin-embedded tissue sections were dewaxed, rehydrated, and incubated in hydrogen peroxide solution for 30 minutes to block endogenous peroxidase activity. Antigen retrieval was carried out by pressure cooker treatment in citrate buffer (pH 6.0) for 40 minutes. Sections were incubated with primary antibody using the conditions specified in Supplemental Table 3 . Secondary antibody (DAKO EnVision+ System-HRP Labelled Polymer anti-mouse, catalog K4001) was applied for 30 minutes, followed by DAB for 5 minutes. Positive controls included breast cancer tissue, fallopian tube, testis, and endometrioid carcinoma.
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