The paraffin‐embedded tissues were subjected to antigen retrieval by microwaving in 0.01 M sodium citrate (pH 6) for 10 min after deparaffinization, hydration and endogenous peroxidase activity elimination, and then incubated overnight at 4 °C with antibody against CD8 (1:100, ab101500, Abcam, Cambridge, UK) or PD1 (1:300, ab137132, Abcam, Cambridge, UK). Subsequently, the slides were incubated with the corresponding secondary antibodies (Zhongshan Goldenbridge Biotechnology, Beijing, China). The number of CD8+ and PD1+ T cells in five nonoverlapping high-power fields (400×) was counted and the average value was calculated.
Ab137132
Manufactured by Abcam
Sourced in United Kingdom, United States, Denmark
Ab137132 is a lab equipment product. It is designed for laboratory use. The core function of this product is to [description not available].
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Lab products found in correlation
Ab205921, by Abcam (4 mentions)
Ab93278, by Abcam (3 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ms457s, by Abcam (2 mentions)
Opal 7 color fluorescence immunohistochemistry ihc kit, by PerkinElmer (2 mentions)
Hrp broad spectrum superpicture polymer detection kit, by Thermo Fisher Scientific (2 mentions)
M361929 2, by Agilent Technologies (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab101500, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Ab130039, by Abcam (1 mentions)
Epr4877, by Abcam (1 mentions)
Eclipse ci l photographic microscope, by Nikon (1 mentions)
Media cybemetics software, by Nikon (1 mentions)
Ab6672, by Abcam (1 mentions)
Ab699, by Abcam (1 mentions)
Ab2406, by Abcam (1 mentions)
Ab190958, by Abcam (1 mentions)
Ab34843, by Abcam (1 mentions)
Superpicture 3rd gen ihc detection kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using ab137132
1
Quantifying CD8+ and PD1+ T Cells
2
Immunohistochemical Analysis of PD-L1, CD8, and HPV in Tumor Tissues
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Tumor sections were freshly cut to 4 mm and dipped in 4% formalin for 24 hours. Tumor sections were then embedded and cut into slices. With antigen repaired, PD-L1 immunohistochemistry (rabbit anti-human PD-L1 monoclonal, 1:200,Cell Signaling 13684; rabbit anti-human CD8 monoclonal 1:100, Abcam ab93278; mouse anti-human HPV16/18 monoclonal stoste, Abcam ab51931; rabbit anti-human PD-1 monoclonal 1:50, Abcam ab137132.rabbit anti-human IgG monoclonal 1:500, Abcam ab172730) was performed and the antibody was incubated for one night at 4℃.
PD-L1, CD8 and HPV expression was evaluated on tumor tissues. Tumor infiltrating immune cells were not identified in all cases. The proportion of PD-L1-positive cells was estimated as the percentage of total tumor cells; tumor cells typically showed membranous staining with a variably component of cytoplasmic staining.
The standard for evaluation scale of marks of the situation of dye is below: The staining intensity of immunohistochemistry was divided into 4 levels: negative, weakly positive, moderate positive, strong positive. We scored negative as 0; weakly positive as 1; moderate positive as 2; strong positive as 3.
The evaluation of the specimens was scored as the following principle:
PD-L1, CD8 and HPV expression was evaluated on tumor tissues. Tumor infiltrating immune cells were not identified in all cases. The proportion of PD-L1-positive cells was estimated as the percentage of total tumor cells; tumor cells typically showed membranous staining with a variably component of cytoplasmic staining.
The standard for evaluation scale of marks of the situation of dye is below: The staining intensity of immunohistochemistry was divided into 4 levels: negative, weakly positive, moderate positive, strong positive. We scored negative as 0; weakly positive as 1; moderate positive as 2; strong positive as 3.
The evaluation of the specimens was scored as the following principle:
3
Immunohistochemical Evaluation of PD-1 and PD-L1 in Tumors
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For mice, tumor sections and commercial antibodies (Abcam, Shanghai, China, ab130039) were used for IHC staining. For patients, tumor tissue samples obtained via core needle biopsy samples obtained from primary lesions were used for IHC staining. Rabbit monoclonal antibodies against PD-L1 (28-8, Abcam, Shanghai, China, AB205921) and PD-1 (EPR4877 (2 (link)), Abcam, Shanghai, China, ab137132) were used for IHC analysis. The reaction was visualized using SignalStain Boost IHC Detection Reagent (HRP, Rabbit). Tumor expression of PD-1 or PD-L1 was considered positive when membrane staining was observed. The following semiquantitative scoring method (6-step scoring system, ‘Cologne Score’) was used for PD-1 and PD-L1: 0 (<1%), 1 (≥1% and <5%), 2 (≥5% and <10%), 3 (≥10% and <25%), 4 (≥25% and <50%), 5 (≥50% and <75%) (17 (link), 18 (link)). Tumors with a score ≥2 were graded as relatively high expression, and tumors with a score ≤1 were graded as having relatively low expression (a cut-off value of 5%). An Eclipse Ci-L photographic microscope (Nikon, Japan) and Media Cybemetics software (USA) were used to analyze tumoral expression of PD-1 and PD-L1. Two experienced pathologists were consulted to ensure the accuracy of semi-quantitative scoring approach used.
4
Histopathological Analysis of Mouse Livers
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Mouse livers were collected at the specified time points, fixed with 10% neutral buffered formalin and embedded in paraffin for processing into 4 micron sections. Sections were rehydrated and stained with Haematoxylin and Eosin (H&E) (Thermo Scientific) or Sirius red and Fast green (Sigma) before pathology evaluation. Alternatively, sections were treated with heat mediated antigen retrieval with pH6 sodium citrate or pH9 Tris-EDTA buffer before staining with primary antibodies. Primary antibodies used in this study include anti-HSA (ab2406, ab19180 Abcam), anti-CD3 (ab699, Abcam), anti-CD20 (555677, BD), anti-CD68 (ab955, Abcam), anti-PD1 (ab137132, Abcam), anti-β-catenin (610154, BD), anti-glutamine synthetase (MAB302, Millipore), anti-LFABP (ab190958, Abcam), anti-IL6 (ab6672, Abcam) and anti-IL10 (ab34843, Abcam).
Immunohistochemistry staining was performed using the Superpicture 3rd Gen IHC Detection Kit (879673, Life Technologies) while immunofluorescence staining was done using anti-mouse IgG, anti-rabbit IgG or anti-Goat IgG secondary antibodies conjugated to Alexafluors 488 or 647 (Life Technologies). Images were captured using an Olympus upright confocal microscope or a Zeiss Axioscan Z1 slide scanner.
Immunohistochemistry staining was performed using the Superpicture 3rd Gen IHC Detection Kit (879673, Life Technologies) while immunofluorescence staining was done using anti-mouse IgG, anti-rabbit IgG or anti-Goat IgG secondary antibodies conjugated to Alexafluors 488 or 647 (Life Technologies). Images were captured using an Olympus upright confocal microscope or a Zeiss Axioscan Z1 slide scanner.
5
Immunohistochemical Analysis of PD-1/PD-L1
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Immunohistochemical (IHC) staining was performed according to standard protocol. Briefly, paraffin-embedded samples were cut into 4 μm sections and placed on polylysine-coated slides. Paraffin sections were baked overnight at 58 °C, de-paraffinized in xylene, rehydrated through graded ethanol, quenched for endogenous peroxidase activity in 0.3 % hydrogen peroxide at 37 °C for 15 min, and processed for antigen retrieval by high pressure cooking in citrate antigen retrieval solution (pH = 6.0) for about 10 min for PD-L1 and EDTA antigen retrieval solution (pH = 8.0) for about 4 min for PD-1. Sections were incubated at 37 °C for 1.5 h with rabbit monoclonal antibodies against PD-1 (1:100, ab137132, Abcam, Cambridge, MA, USA) and PD-L1 (1:50, ab174838, Abcam, Cambridge, MA, USA) in a moist chamber. Immunostaining was performed using the EnVision+System-HRP (AEC) (K4005, Dako, Glostrup, Denmark), which resulted in a brown-colored precipitate at the antigen site. Subsequently, sections were counterstained with hematoxylin (Sigma-Aldrich, St Louis, MO, USA) and mounted in a non-aqueous mounting medium. All runs included a no primary antibody control.
6
Immunofluorescence Analysis of Liver Biomarkers
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For immunofluorescence analysis, tissues were stained with CD8 antibody (ab60076, abcam, Cambridge, USA), PD1 antibody (ab137132, abcam), SALL4 antibody (ab29112, abcam), hepatocyte specific antigen antibody (GTX73779, Genetex), CD45 antibody (MA5-17687, Thermo Scientific, Waltham,USA) and PD-L1 (ab205921, abcam) followed by Alexa Fluor 647 conjugated goat anti-rat IgG (A-21247), Alexa Fluor Plus 488 conjugated goat anti-rabbit IgG (A-32731) and Alexa Fluor Plus 555 conjugated donkey anti-mouse IgG (A-32727) (Thermo Scientific). Images were acquired on a Zeiss 710 Meta multi-photon confocal microscope (Zeiss, Oberkochen, Germany). To assess the immunostaining quantification, we analyzed the slides via an image analysis workstation (Image Pro Plus 6.0, Media Cybernetics). Mouse liver tissues were collected and embedded in OCT. Intrahepatic HBsAg, Pd-l1, or Sall4 expression was visualized by immunohistochemical staining with rabbit anti-mouse HBs Ab (Genetech, Shanghai, China) or rabbit anti-mouse Pd-l1 Ab (eBioscience, San Diego, CA, USA), or anti-Sall4 Ab (abcam, cat#57577) followed by Envision System HRP detection staining (Genetech, Shanghai, China) performed according to the manufacturer’s protocol. Liver sections were stained with hematoxylin. Images were taken with an OLYMPUS microscope.
7
Immunohistochemical Analysis of Tumor Markers
8
Immunohistochemistry Analysis of PD-L1, CD3, and PD1
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A tissue microarray (TMA) was constructed by Shanghai Biochip Co. Ltd. (Shanghai, China), as described previously 29 (link). Immunohistochemistry (IHC) was performed with Leica BOND-MAX system (Wetzlar, Germany), as described previously 30 (link). Primary antibodies used were: monoclonal rabbit anti-human PD-L1 (dilution 1:100; #SP142, GeneTech Co. Ltd., Shanghai, China), monoclonal rabbit anti-human CD3 (dilution 1:500; #NCL-L-CD3-565, Leica, Cambridge, UK), and monoclonal rabbit anti-human PD1 antibody (dilution 1:500; #ab137132, Abcam, Cambridge, UK). Images were captured with Leica Q Win Plus v3 software (Leica Microsystems Imaging Solutions, Cambridge, UK). ICC diagnosis was validated by H&E staining.
9
Multiplex Immunofluorescence for Immune Profiling
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Multiplexed immunofluorescence staining was performed using an Opal 7-Color Fluorescence Immunohistochemistry (IHC) Kit (PerkinElmer, Waltham, MA, USA), according to the manufacturer’s protocol with the following primary antibodies: CD8 (1:800, ab93278; Abcam), PD-1 (1:800, ab137132; Abcam), D240 (1:500, M361929-2; Dako, Glostrup, Denmark), and PD-L1 (5 μg/mL, ab205921; Abcam). After deparaffinization, the sections were microwaved in antigen retrieval buffer for 45 s at 100°C, washed and blocked for 10 min at 25°C, and incubated with each primary antibody. Next, the slides were incubated with an HRP-broad spectrum SuperPicture Polymer Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated with Opal fluorochromes (Opal520, Opal570, Opal 620, and Opal 690), diluted 1:150 in amplification buffer (all provided by the Opal 7-Color Fluorescence IHC Kit) for 10 min at 25°C. Finally, the slides were microwave treated with AR6 buffer, incubated with a working 4′,6-diamidino-2-phenylindole (DAPI) solution (provided in the Opal 7-Color Fluorescence IHC Kit) for 5 min at 25°C, and mounted with ProLong Diamond Antifade Mounting Medium (Life Technologies). Images were obtained using a Zeiss LSM 880 Confocal Laser-Scanning Microscope (Jena, Germany).
10
Immunohistochemical Analysis of Immune Markers
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Immunohistochemical staining was performed on 10% formalin-fixed and paraffin-embedded tissues. Antibodies of CD16 (Anti-CD16: Abcam, ab246222, at a dilution of 1:100), CD56 (Anti-NCAM1: Abcam, ab75813, at a dilution of 1:100), IL-6 (Anti-IL6: Bioworld, MB9296, at a dilution of 1:50), and PD-1 (Anti-PD-1: Abcam, ab137132, at a dilution of 1:250) were used. By the way, in the stained sections PD-1 was negative, we predicted that there is not any PD-1 expression in the selected samples, so it was not reflected in the text. The mean gray value method was used for the quantification of immunohistochemistry and immunofluorescence by ImageJ (NIH 64-bit Java 1.8.0).
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