AR-V7 and AR-FL IHC was performed as previously described (30 (link), 31 (link)). Briefly, AR-V7 IHC was performed using recombinant rabbit monoclonal anti–AR-V7 antibody (Clone RM7, RevMAb Biosciences). Biopsies were first deparaffinized before antigen retrieval by microwaving (in Tris/EDTA buffer, pH 8.1) for 18 minutes at 800 W, and anti–AR-V7 antibody (1:500 dilution in Dako REAL diluent, Agilent Technologies) was incubated with tissue for 1 hour at room temperature. After washes, bound antibody was visualized using Dako EnVision Detection System (Agilent Technologies). Sections were counterstained with hematoxylin. Cell pellets from 22Rv1 (positive) and PC3 (negative) were used as controls. Rabbit IgGs were used as a further negative control. AR-FL IHC was performed using mouse monoclonal anti-AR antibody (AR441, Agilent Technologies). Biopsies were first deparaffinized before antigen retrieval using pH 8.1 Tris/EDTA solution heated in a water bath, and anti-AR antibody (1:1,000 dilution in Dako REAL diluent, Agilent Technologies) was incubated with tissue for 1 hour at room temperature. After washes, bound antibody was visualized using Dako EnVision Detection System (Agilent Technologies). Sections were counterstained with hematoxylin. Cell pellets from VCaP (positive) and PC3 (negative) were used as controls. Mouse IgGs were used as a further negative control.
Dako envision detection system
Manufactured by Agilent Technologies
Sourced in Denmark, United States, Japan
The DAKO EnVision Detection System is a laboratory equipment product used for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. It functions as a detection system, enabling the visualization of target proteins or nucleic acids in tissue samples.
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Lab products found in correlation
Protein block, by Leica Biosystems (1 mentions)
Anti pd l1, by Cell Signaling Technology (1 mentions)
Clone d9m2l, by Cell Signaling Technology (1 mentions)
Optimas version 6, by Media Cybernetics (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Anti pold1, by Proteintech (1 mentions)
Tyr416, by Cell Signaling Technology (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Nanozoomer, by Hamamatsu Photonics (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Anti glutamine synthetase, by BD (1 mentions)
Anti β catenin, by BD (1 mentions)
Dako real diluent, by Agilent Technologies (1 mentions)
Ar441, by Agilent Technologies (1 mentions)
Protease 1, by Roche (1 mentions)
S9e microscope, by Leica camera (1 mentions)
Sybr green dye, by Merck Group (1 mentions)
Rabbit anti hbcag polyclonal antibody, by Agilent Technologies (1 mentions)
Clone mib 1, by Agilent Technologies (1 mentions)
22 protocols using dako envision detection system
1
Immunohistochemical Detection of AR-V7 and AR-FL
2
Histological Analysis of Tissue Samples
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All specimens were fixed with formalin, embedded by paraffin and cut into 4 μm sections. Tissue slices were subjected to hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) staining using the two-step method of Dako Envision™ Detection System (DakoCytomation, Glostrup, Denmark). Briefly, tissue slices were incubated with primary antibodies (listed in Additional file 2 : Table S2) after deparaffinized, rehydrated and antigen retrieval routinely. All stained slides were reviewed independently by two pathologists. Semi-quantitative analysis of IHC results was described previously [26 (link)]. In brief, IHC score was calculated by multiplying staining intensity score (score of 0, 1, 2 and 3 represented negative, weak-positive, moderate-positive and strong-positive, respectively) and the score of relative positive-staining area (score of 0, 1, 2, 3, and 4 indicated positive-staining areas of 0–5%, 6–25%, 26–50%, 51–75% and 76–100%, respectively). IHC score more than 2 was defined as high expression, while the others represented low expression (Additional file 3 : Figure S1).
3
Immunophenotyping of ENKTCL Tissue Samples
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The immunochemistry (IHC) staining was performed on 4 μm FFPE ENKTCL sections by a professional pathologist according to our previous study.34 (link) Briefly, after deparaffinization, rehydration and endogenous peroxidase inactivation, antigen retrieval was performed using EDTA buffer (PH 9.0) in microwave, followed by blocking nonspecific binding with protein block (Novocastra, Newcastle, UK). Then, slides were incubated with primary antibody (anti-PD-L1: cell signaling technology (CST), #13684; anti-VISTA: CST, #54979; anti-B7-H3: CST, #14058; anti-B7-H4: CST, #14572; anti-HHLA2: Sigma-Aldrich, HPA055478; anti-CD8: CST, #85336; anti-Foxp3: Abcam, ab215206) at 4 °C overnight. After incubation with the corresponding second antibody, the slides were visualized with a DAKO EnVision Detection System (Dako). In situ hybridization (ISH) was performed to detect the EBV encoded small RNA (EBER) in FFPE tissue slides as previously described.3 (link)
4
Immunohistochemical Analysis of Immune Checkpoints in Prostate Cancer
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In line with our previous study,30 (link) briefly, formalin-fixed PCa tissue sections were paraffinized in xylene and hydrated in a diluted alcohol series. Then, antigen retrieval was accomplished with sodium citrate buffer (10 mM, pH 6.0) using a microwave for 10 min. After that, the sections were immersed in H2O2 (0.3%) for 15 min to reduce activity of endogenous peroxidase. Following blocking with goat serum, the sections were incubated with corresponding primary antibodies (anti-PD-L1: Cell Signaling Technology (CST) #13684; anti-B7-H3: CST, #14058; anti-HHLA2: clone 566.129 (link); anti-CD8: CST, #85336; anti-Foxp3: CST, #12653) at 4℃ overnight. Then, after washing for three times using phosphate buffered saline (PBS), the sections were subsequently incubated with secondary antibody for 1 hour. Finally, the sections were visualized using a DAKO EnVision Detection System (Dako) and counterstained with hematoxylin.
5
Immunohistochemical Analysis of Immune Markers
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Immunohistochemistry (IHC) staining for HHLA2, PD-L1, CD8, and CD4 was accomplished by a professional pathologist.23–25 (link) After deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inactivation, and blocking non-specific binding, the 4 µM-thick sections were incubated with primary antibodies (anti-HHLA2: Sigma-Aldrich, HPA055478; anti-PD-L1: cell signaling technology, CST #13684; anti-CD8: CST, #85336; anti-CD4: Abcam, ab252199) at 4°C overnight. Then, the slides were incubated with a corresponding secondary antibody and visualized by using a DAKO EnVision Detection System (Dako). Finally, the slides were counterstained with hematoxylin, dehydrated, and cover-slipped.
6
Immunohistochemical Analysis of B7-H3 Expression
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Immunohistochemistry (IHC) staining for B7-H3 was interpreted by two pathologists in a blinded manner. Representative 4-μm-thick, formalin-fixed, paraffin-embedded (FFPE), resected specimens were placed onto the glass slides after deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inactivation, and blocking of nonspecific binding. B7-H3 was stained overnight at 4°C using the D9M2l rabbit monoclonal antibody (1:200, clone D9M2l, catalog 14058s, Cell Signaling Technology, Danvers, MA, United States ). Finally, the reaction products were imaged using a DAKO EnVision Detection System (Dako), and hematoxylin was used for counterstaining.
7
Immunohistochemical Analysis of IL-6 in Renal Cell Carcinoma
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Formalin-fixed, paraffin-embedded human tissues including renal cell carcinoma were processed for immunohistochemical staining with an anti-IL-6 antibody (ROCKLANDTM Gilbertsville, PA). Sections (2 μm thick) were deparaffinized in three changes of xylene, hydrated through a descending series of ethanol. The endogenous peroxidase activity was blocked with methanol containing 0.5% hydrogen peroxide for 20 minutes. Then sections were treated at 4 °C overnight with primary antibodies for IL-6 (1:600 dilution). Subsequent reactions were carried out with the DAKO EnVision Detection System (Dako, Hamburg, Germany) according to the manufacturer's instructions.
8
Immunohistochemical Analysis of Fibrotic Markers
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Detection of proteins, α‐SMA, TGF‐β1, CTGF and MT1‐MMP, were carried out using respective primary antibody and Dako EnVision™ Detection System (K5007; DAKO, Glostrup, Denmark). Briefly, paraffin‐embedded samples were cut into 3‐μm‐thick sections, deparaffinized and rehydrated. After antigen retrieval using Target Retrieval Solution (S2375; DAKO), non‐specific binding was blocked with 10% normal goat serum for 1 h. Sections were then incubated with primary antibodies against α‐SMA (1:200), TGF‐β1 (1:200), CTGF (1:400) and MT1‐MMP (1:40) in dilution buffer overnight at 4°C in a humidified chamber. Slides were counterstained with hematoxylin and examined under a microscope (Olympus AX70; Olympus, Tokyo, Japan). For quantitative analysis, % area of stained region was calculated using image analysis software OPTIMAS version 6.5 (Media Cybernetics, Silver Spring, MD, USA).
9
Immunohistochemical Analysis of TRIM37 in CRC
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Clinical CRC tissues and paired non-cancerous tissues (n=30) were fixed in formalin, embedded in paraffin, and cut into 5-µm-thick consecutive sections. Slides were dewaxed, rehydrated, and the epitope was subsequently retrieved. After blocking the enzymatic activity of endogenous peroxidase, slides were immunolabeled using TRIM37 primary antibody (1:200; cat. no. sc-49548, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, the slides were washed with PBS and incubated with secondary antibody (1:1,000; cat. no. GK500705; Dako; Aligent Technologies, Santa Clara, CA, USA) and 3,3′-diaminobenzidine using the Dako EnVision Detection System (Dako; Aligent Technologies). The slides were then counterstained with hematoxylin.
10
Immunohistochemical Analysis of SOX7 Expression
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The TMA slides were deparaffinized with xylene and then serially rehydrated with ethanol (100, 100, 95, 95, 90, 80 and 70%). Following brief proteolytic digestion using the IHC enzyme antigen retrieval agent (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and peroxidase blocking with 3% H2O2, the slides were incubated at 4°C overnight with a rabbit polyclonal primary antibody against SOX7 (dilution, 1:200; cat. no. sc-20,093; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing with PBS to remove any unbound primary antibodies, the slides were incubated for 30 min with peroxidase-conjugated secondary antibody (HRP-labeled Goat Anti-Rabbit IgG; dilution, 1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology Co., Ltd., Haimen, China) at room temperature. The specifically bound secondary antibody was detected using a DAKO EnVision detection system (Dako Diagnostics; Agilent Technologies, Inc., Santa Clara, CA, USA).
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