Sections of frozen metastatic melanoma tumor tissue were thawed at room temperature (RT) and fixed with pre-cooled Acetone and Ethanol for 10 minutes each. Sections were washed and incubated with blocking buffer (0.1% BSA in 0.1% TBS-T) for 1 hour at RT, prior to primary antibody incubation for 1 hour at RT with one of either 5µg/ml rabbit anti-IL-10 (polyclonal, Abcam), 8µg/ml rabbit anti-TNF-α (TNFA/1500R, Abcam), or 1µg/ml rabbit anti-TGFβ1 (EPR21143, Abcam) in addition to 10µg/ml rat anti-CD3 (CD3-12, Abcam) and mouse anti-CD20 (1/50 dilution, L26, Abcam). Sections were washed and incubated with 10µg/ml cross-adsorbed donkey secondary antibodies (Abcam): anti-rabbit IgG AF594, anti-mouse IgG AF488 and anti-rat IgG AF647. Sections were washed and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Slides were incubated for 24 hours prior to visualization on the Olympus VS120-S6-W slide scanning microscope.
Cd3 12
Manufactured by Abcam
Sourced in United States
CD3-12 is a monoclonal antibody that specifically recognizes the CD3 epsilon subunit of the T cell receptor complex. It is commonly used in flow cytometry and immunohistochemistry applications to identify and characterize T cells.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Vs120 s6 w slide scanning microscope, by Olympus (1 mentions)
Epr21143, by Abcam (1 mentions)
Anti rabbit igg af594, by Abcam (1 mentions)
Goat anti rat igg h l, by Abcam (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
3 protocols using cd3 12
1
Multiplex Immunofluorescence Profiling of Melanoma
2
Quantifying CD3+ T Cell Infiltration
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CD3+ cell infiltration was assessed on spleen and liver graft sections as follows: Tissue sections were heated for 15 min in EDTA (pH 8.0) in a microwave for antigen retrieval, incubated in 3% hydrogen peroxide for 30 min to eliminate endogenous peroxidase activity, and blocked in normal goat serum. Then the sections were incubated overnight at 4 ℃ with a rat anti-CD3 antibody (CD3-12, Cat# ab11089, RRID: AB_2889189; Abcam, Cambridge, MA, United States) and incubated with a secondary antibody (Goat Anti-Rat IgG H&L, Cat# ab97057, RRID: AB_10680316; Abcam, Cambridge, MA, United States) or 30 min at room temperature the next day. Finally, the sections were stained with freshly prepared diaminobenzidine solution and counterstained with hematoxylin. ImageJ software was applied to 200 × images to quantify the CD3-positive fields.
3
Immunohistochemical Analysis of Kidney Immune Cells
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Paraffin-embedded kidney sections were deparaffinized and rehydrated, and antigen retrieval was done, followed by washing and staining with rat antihuman CD3 (1:200; CD3-12; Abcam), mouse antihuman CD4 (1:10; 4B12; Thermo Fisher Scientific), and rabbit antihuman GPR56 (1:250; polyclonal; Thermo Fisher Scientific) antibodies.
After overnight incubation, slides were washed and stained with secondary antibodies: antirat Alexa fluor 637, antimouse Alexa fluor 488, and antirabbit Alexa fluor 594 (Thermo Fisher Scientific), respectively, for 1 hour. The slides were simultaneously stained with 4′,6-diamidino-2-phenylindole (1:10,000; Sigma Aldrich), and antifade mounting medium was added after completion of the staining (ProLong Gold from Invitrogen). The slides were viewed under a 4-color confocal microscope (LSM900; Zeiss Inc.). Healthy tonsils and colon sections were used as negative control for GPR56 staining on CD4+T cells.
After overnight incubation, slides were washed and stained with secondary antibodies: antirat Alexa fluor 637, antimouse Alexa fluor 488, and antirabbit Alexa fluor 594 (Thermo Fisher Scientific), respectively, for 1 hour. The slides were simultaneously stained with 4′,6-diamidino-2-phenylindole (1:10,000; Sigma Aldrich), and antifade mounting medium was added after completion of the staining (ProLong Gold from Invitrogen). The slides were viewed under a 4-color confocal microscope (LSM900; Zeiss Inc.). Healthy tonsils and colon sections were used as negative control for GPR56 staining on CD4+T cells.
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