Immunohistochemical analyses were performed using a standard protocol detailed previously.35 36 (link) Antigens were retrieved in 10 mM citrate (pH 6) (Sigma Aldrich) or EDTA (pH 9) (Zytomed Systems, Berlin, Germany) buffer. The primary antibodies used in the present study were as followed: anti-HMGB1 (clone 1D5, Abnova, Taipei City, Taiwan), anti-cleaved caspase 3 (clone C92-605, BD Biosciences), anti-CD31 (ab28364, Abcam, Cambridge, UK), anti-CD3 (clone 2GV6, Ventana Medical Systems, Tucson, Arizona, USA), anti-Foxp3 (clone 236 A/E7; eBioscience, San Diego, California, USA), anti-CD68 (clone KP-1, Ventana Medical Systems), anti-CD206 (clone E2L9N, Cell Signaling Technology, Danvers, MA, USA), anti-PD-1 (clone NAT105, Abcam), anti-human PD-L1 (clone 28–8, Abcam) and anti-mouse PD-L1 (orb10162, Biorbyt, Cambridge, UK). The secondary reaction was performed using the mouse or rabbit Envision+system (Dako, Glostrup, Denmark) according to the manufacturer’s recommendations. Positive cells were visualized using a 3,3’-diaminobenzidine (DAB) substrate (Cell Signaling Technology). Mouse or rabbit control IgGs (Santa Cruz Biotechnology, Santa Cruz, California, USA) were used as negative controls.
Anti pd 1 clone nat105
Manufactured by Abcam
Sourced in United States, Azerbaijan
Anti-PD-1 (clone NAT105) is a monoclonal antibody that specifically binds to the PD-1 protein. PD-1 is a cell surface receptor that plays a key role in regulating T-cell activity and immune response.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 clone c8 144b, by Agilent Technologies (2 mentions)
Anti foxp3 clone 236 a e7, by Thermo Fisher Scientific (1 mentions)
Anti cd68 clone kp 1, by Roche (1 mentions)
Anti cd31 ab28364, by Abcam (1 mentions)
Anti cd3 clone 2gv6, by Roche (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Dako autostainer plus, by Agilent Technologies (1 mentions)
Anti foxp3 clone 236a e7, by Abcam (1 mentions)
Anti cd3 clone ln10, by Leica (1 mentions)
Anti cd163 clone 10d6, by Leica (1 mentions)
Anti pd l1 clone sp263, by Roche (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Opal 7 color fluorescence immunohistochemistry kit, by Akoya Biosciences (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti pd 1 clone nat105
1
Comprehensive Immunohistochemical Analyses
2
Immune Checkpoint and Infiltration Analysis in CLL and RS
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Expression of the immune checkpoint molecules in CLL and RS lymph node samples was analyzed by IHC staining using antibodies specific for PD-L1 and PD1. Immune cell infiltration in CLL and RS lymph node samples was assessed by IHC staining using antibodies specific for CD3, CD8, FOXP3, and CD163. IHC staining was done on 4-μm FFPE sections on DAKO Autostainer Plus (Agilent, Santa Clara, CA) using standard protocol17 (link),20 (link). The antibodies used include anti-PD-L1 (clone SP263, Ventana Medical Systems, Inc., Tucson, AZ), anti-PD1 (clone NAT105, Abcam, Inc., Cambridge, MA), anti-CD3 (clone LN10, Leica Biosystems, Newcastle Upon Tyne, UK), anti-CD8 (clone C8/144B, Dako, Carpenteria, CA), anti-FOXP3 (clone 236A/E7, Abcam, Inc., Cambridge, MA), and anti-CD163 (clone10D6, Leica Biosystems, Newcastle Upon Tyne, UK). IHC images were taken using whole slide imaging technology with MoticEasyScan Pro (Motic digital pathology, San Francisco, CA), and saved in tagged image file format. Percentage of expression for each individual antigen was calculated by dividing number of cells with positive staining by number of total cells in the image using the Image-Pro premier 3D 9.1.4 software (Media Cybernetics, Silver Spring, MD).
3
Multiplex Immunofluorescence Staining Workflow
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Multiplex immunofluorescence staining was performed using the OPAL 7-color fluorescence immunohistochemistry kit (Akoya Biosciences, Marlborough, MA). Tissue sections were deparaffinized in xylene and rehydrated with serial passage through graded ethanol. Endogenous peroxidase was blocked with 0.3% H 2 O 2 /methanol for 10 minutes. Antibodies used included anti-PD-1 (clone NAT105, Abcam), anti-CD4 (clone 4B12, NeoMarkers, Portsmouth, NH), anti-CD8 (clone C8/144B, Dako), anti-CD103 (clone EPR4166 (2), Abcam), anti-SOX10 (clone EP268, Monosan, Uden, the Netherlands), and anti-CD3 (Dako). Steps were repeated for each primary antibody. Heat-induced antigen retrieval was performed for 10 minutes at 95°C in Tris-EDTA pH 9.0 buffer. Slides were washed in TBST and blocked with blocking/Ab diluent for 10 minutes, before being stained with primary antibody for 60 minutes at room temperature. Afterward, slides were incubated with Polymer HRP Ms + Rb for 10 minutes. Next, slides were incubated with Opal fluorochromes (Opal 520, Opal 620, Opal 480, Opal 570, Opal 690, and Opal 780) for 10 minutes. Finally, DAPI working solution was applied for 5 minutes, and slides were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA).
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