Next, Immunohistochemistry (IHC) staining was conducted on these tissue slides. The primary antibodies used in the research were as follows: anti-ACE2 (1:3000 dilution, Cat. ab15348, Abcam, Cambridge, UK), anti-CD8 (Ready-to-use, Cat. PA067, Abcarta, Suzhou, China), and anti-PD-L1 (Ready-to-use, Cat. GT2280, GeneTech, Shanghai, China). Antibody staining was visualized with DAB and hematoxylin counterstain, and stained sections were scanned using Aperio Digital Pathology Slide Scanner. All stained sections were independently evaluated by two independent pathologists. For semi-quantitative evaluation of ACE2 and PD-L1 staining, the immunoreactivity score (IRS) was applied as previously described [27 (link)]. For CD8 staining, infiltration level was assessed by estimating the percentage of cells with strong intensity of membrane staining in the stroma cells. In addition, tumors were demarcated into three phenotypes based on the spatial distribution of CD8+ T cells, including the inflamed, the excluded, and the deserted phenotypes [28 (link)].
Anti cd8
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-CD8 is a lab equipment product that can be used to detect and quantify the presence of CD8 proteins, which are found on the surface of certain types of T cells. The product functions by binding to the CD8 protein, allowing for its identification and measurement. No further interpretation or extrapolation on the intended use of this product is provided.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4, by Abcam (18 mentions)
Anti cd3, by Abcam (13 mentions)
Anti ki67, by Abcam (5 mentions)
Anti cd163, by Abcam (5 mentions)
Anti cd19, by Abcam (5 mentions)
Anti pd l1, by Abcam (4 mentions)
Anti cd68, by Abcam (4 mentions)
Anti cd15, by Abcam (4 mentions)
Anti pd l1, by Cell Signaling Technology (4 mentions)
Anti pd 1, by Cell Signaling Technology (4 mentions)
Anti panck, by Abcam (4 mentions)
Anti pd 1, by Abcam (3 mentions)
Anti foxp3, by Abcam (3 mentions)
Anti cd18, by Abcam (3 mentions)
Anti cd7, by Abcam (3 mentions)
Anti cd20, by Abcam (3 mentions)
Opal automation mif detection kit, by Akoya Biosciences (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Vectra polaris automated quantitative pathology imaging system, by Akoya Biosciences (3 mentions)
Anti epcam, by Abcam (2 mentions)
61 protocols using anti cd8
1
Immunophenotyping of Tissue Samples
2
Immunohistochemical Analysis of Brain Tissue
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4% PFA‐fixed tissues were paraffin embedded and sectioned into serial slices of 5 μm thickness. After permeabilization, antigen retrieval, and blocking, the brain slices were incubated with primary antibodies anti‐Ki‐67 (Abcam, ab 16667, 1:1000), anti‐CD8 (Abcam 1:1500), and cleaved caspase‐3 (CST Asp175 1:2500) diluted with 1% goat serum (Vector Laboratories) in PBS overnight. The slices were then incubated in the secondary antibody for 1 h and stained with the ABC HRP kit (Vectastain). After washing, the bound complex was visualized by incubating it with DAB (3,3‐diaminobenzidine). Images were obtained by Nikon‐Eclipse Ti. Image analysis was performed using Image J 1.51j8 with IHC Profiler to calculate the positive stained areas and to count the number of positive cells.
3
Histological Evaluation of Liver Tumors in Mice
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Formalin-fixed and paraffin-embedded tissues sectioned to 4 μm were used for histological evaluation of liver tumors in a mouse model. Hematoxylin and eosin (H&E) staining was performed for each sample. For IHC, tissue slides were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions (100%, 95%, and 70%). Subsequently, the slides were subjected to antigen retrieval by microwaving in a citric acid solution for 15 min. The primary antibodies anti-hepatocyte (Abcam, Cat#ab75677), anti-Epcam (Abcam, Cat#ab213500), anti-CD3 (Abcam, Cat#ab16669), anti-Ly6G (Servicebio, Cat#GB11229), anti-CD8 (Abcam, Cat#ab217344), anti-F4/80 (CST, Cat#70076), anti-Asma (Servicebio, Cat#BM0002), and anti-CD31 (Servicebio, Cat#GB113151) were used. Subsequently, the slides were incubated with secondary antibodies (1:1, 100 μL for each slide; HRP-anti-rabbit IgG, ZSGB, Cat#PV-6001, or HRP-anti-mouse IgG, ZSGB, Cat#PV-6002) for 10 min at room temperature. Multispectral images were scanned with a ZEISS AXIOSCAN 7 instrument. Cells of interest were quantified in Halo v3.4 (Indica Labs) or QuPath v0.2.0. Each section was evaluated by 2 or 3 experienced pathologists.
4
Quantifying Immune Cell Profiles in Tumor, Intestine, and Spleen Tissues
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Immunohistochemical (IHC) staining was performed on tumor, intestinal and spleen tissues to quantify changes in immune cells (CD4 + T, CD8 + T, MPO-positive neutrophils, and F4/80-positive macrophages). The sections were then deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide for 30 min at room temperature. Nonspecific binding was reduced by incubation with 10 % normal goat serum for 30 min. The sections were then incubated with rabbit antibody anti-CD4 (CST, Massachusetts, Cat. 25229, 1:125 dilution), anti-CD8 (Abcam, Cambridge, Cat. ab209775, 1:100 dilution), anti-MPO (Abcam, Cambridge, Cat. ab208670, 1:2000 dilution), or anti-F4/80 (CST, Massachusetts, Cat. 70076; 1:500 dilution) overnight at 4 °C. This was followed by a 45-min incubation with secondary antibodies. Immunoreactivity was revealed using the diaminobenzidine chromogen reaction. The detailed information on primary and secondary antibodies used for IHC staining is summarized in the Supplementary TableA2.
The IHC sections were scanned using a KF-PRO-020 whole slide scanner (KFBIO, Ningbo, China) and then analyzed using the HALO image analysis platform (Indica Labs, Albuquerque, NM, USA), as described in our previous publication [4] (link).
The IHC sections were scanned using a KF-PRO-020 whole slide scanner (KFBIO, Ningbo, China) and then analyzed using the HALO image analysis platform (Indica Labs, Albuquerque, NM, USA), as described in our previous publication [4] (link).
5
Isolation and Flow Cytometry of Immune Cells
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Briefly, peripheral elbow venous blood (5 ml) was obtained from all patients within 24 h after admission. The samples were collected in tubes with heparin and added with 5 ml phosphate buffer (PBS) and then centrifuged at 1500 g for 10 min. The cell layer was then collected, added with 5 ml PBS, following with centrifugation at 1200 g for 5 min. After removing the supernatant and washing with PBS, the samples were further centrifuged at 1000 g for 2 min to obtain the peripheral blood mononuclear cells. The cells with density of 1 × 106/ml were maintained in RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, USA).
For measurement of PD-1+ and LAG-3+ T cells, flow cytometry was performed as described elsewhere [17 (link), 18 (link)]. Antibodies used in this study included anti-CD4, anti-CD8, anti-PD-1 and anti-LAG-3 (all purchased from Abcam, USA). The measurement was conducted on a FACS Calibur flow cytometry analyzer (BD Biosciences) using Diva software (version 6.1, BD Pharmingen).
For measurement of PD-1+ and LAG-3+ T cells, flow cytometry was performed as described elsewhere [17 (link), 18 (link)]. Antibodies used in this study included anti-CD4, anti-CD8, anti-PD-1 and anti-LAG-3 (all purchased from Abcam, USA). The measurement was conducted on a FACS Calibur flow cytometry analyzer (BD Biosciences) using Diva software (version 6.1, BD Pharmingen).
6
Immunohistochemical Profiling of Mouse Brain
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Brain tissues from mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 µm-thick segments. After heat-induced antigen epitope retrieval, slides were blocked with PBS containing 3% normal serum and incubated overnight at 4 °C with primary antibodies as follows: anti-CD68, anti-CD86 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD4, anti-CD8, anti-Iba-1, and anti-TSPO (Abcam, Cambridge, UK). The slides were then washed with PBS and incubated with biotinylated secondary antibodies (anti-goat for CD68; anti-rabbit for CD4, CD8, and Iba-1; all from Santa Cruz Biotechnology). The slides were then treated with avidin-biotin solution (Vector Laboratories, Burlingame, CA, USA) for 1 h. The antigenic signal was developed using DAB (3, 3 -diaminobenzidine) substrate (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer's instructions. Finally, the slides were counterstained with hematoxylin and mounted with the Permount Mounting Medium (Thermo Fisher Scientific, Fair Lawn, NJ, USA).
7
Comprehensive Immunohistochemistry Protocol
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Immunohistochemistry (IHC) was performed using standard procedures [20 (link)]. Tissue sections were incubated with the following antibodies: anti‐KMT2C (1:1,000), anti‐H3K4me1 (1:1,000), anti‐H3K4me3 (1:1,000), anti‐CD163 (1:500), anti‐CD3 (1:200), anti‐CD4 (1:1,000), anti‐CD8 (1:2,000), and anti‐PD‐L1 (1:500) antibodies (all from Abcam, Cambridge, UK). Details are given in Supplementary materials and methods.
8
Multiplex Immunohistochemistry of Pancreatic Cancer
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Fluorescent mIHC was performed in serial sections of FFPE tumor tissue from each PDAC patient using the Opal 7-Colour Manual IHC Kit (PerkinElmer Hopkinton, Massachusetts, USA) according to the manufacturer’s protocol and as described in our previous article. In short, sections experienced primary antibodies including Rabbit monoclonal antibodies anti-CD68 (1:500, Abcam, Cambridge, MA, USA), anti-CXCR2 (1:500, Abcam), anti-CD206 (1000, Abcam), anti-CXCL1 (1:200, Abcam), anti-CXCL5 (1:200, Abcam), anti-CXCL8 (1:500, Abcam) and anti-CD8 (1:2000, Abcam), followed by HRP-conjugated secondary antibody and fluorescent dyes (Opal520, Opal570, Opal620, Opal690, and DAPI). Stained sections were scanned and processed by a Vectra PolarisTM Automated Quantitative Pathology Imaging System (AKOYA Biosciences, PerkinElmer, Massachusetts, USA).
9
Immunohistochemical Analysis of FMNL3, PD-L1, and CD8
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IHC staining was conducted directly on the TMA with standard procedures. The primary antibodies used in the current studies were as follows: anti-FMNL3 (1:2000 dilution, Cat. ab222797, Abcam, Cambridge, UK), anti-PD-L1 (1:100 dilution, Cat.ab237726, Abcam, Cambridge, UK), and anti-CD8 (Ready-to-use, Cat. PA067, Abcarta, Suzhou, China). Antibody staining was visualized using diaminobenzidine (DAB) and hematoxylin counterstain, and the stained TMA was scanned using Aperio Digital Pathology Slide Scanners. For the semiquantitative assessment of FMNL3 and PD-L1 staining, the immunoreactivity score (IRS) was used according to the previous description.27 (link),28 (link) For semiquantitative evaluation of CD8 staining, the proportion of CD8+ cells was assessed by estimating the percentage of cells with strong intensity of membrane staining in the stroma cells.29 (link)
10
Immunohistochemistry of Immune Markers
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The primary antibodies used in immunohistochemistry (IHC) for tissue slides were used for IHC of tissue slides: anti-IFNAR1 (Abclonal, 1:200 dilution), anti-CD4 (Abcam, 1:200 dilution), anti-CD8 (Abcam, Cat# ab93278, 1:500 dilution), anti-FOXP3 (CST, 1:200 dilution), anti-OAS1 (CST, 1:100 dilution), and anti-TGFBR1 (Abcam, 1:400 dilution). The slices were deparaffinized and rehydrated, pretreated with a citric acid antigen retrieval solution (pH = 6.8), and rinsed in PBS. The sections were blocked in 2% goat serum and incubated with the primary antibody overnight at 4°C. The streptavidin-peroxidase method (ZSGG-BIO, CAT# PV9001) was used to show the levels of stained proteins. The number of CD4, CD8, and FOXP3 cells were calculated using K-Viewer V1 system (Version, Code 1.5.3.1, KONFOONG BIOTECH INTERNATIONAL CO., LTD). The levels of OAS1, IFNAR1, and TGFBR1 were scored as follows: 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The intensity of positive staining was classified into four scales as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The levels were semiquantitatively determined as percentages multiplied by intensity.
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