For Hematoxylin and Eosin (H&E) staining, spleens were fixed overnight in formaldehyde/zinc fixative, paraffin embedded, sectioned and stained using a standard H&E protocol. For immunofluorescence, spleens were snap frozen on dry ice in OCT (Tissue-Tek) and stored at −80°C. 6-µm frozen sections were cut, fixed with 4% paraformaldehyde for 20 min, blocked with 10% FBS, and stained overnight at 4°C with either guinea pig antibody to LCMV (1:1,000 dilution), rabbit anti-mouse CD3 antibody (Abcam, 1:200), rat anti-mouse MOMA-1 antibody (Abcam, 1:200) or rat anti-ER-TR7 antibody (1:250, Abcam). Tissues were washed, incubated at 1 h with Alexa Fluor 488–conjugated antibody to guinea pig IgG (1:200, Invitrogen), Alexa Fluor 568-conjugated anti-rabbit IgG (1:200, Invitrogen) and Alexa Fluor 488- or 568-conjugated antibody to rat IgG (1:200, Invitrogen), washed, stained with 4,6-diamidino-2-phenylindole (DAPI) and mounted using Prolong Gold mounting medium (Life Technologies). Images were taken with a Zeiss Axiovert S100 immunofluorescence microscope fitted with an automated xy stage, an Axiocam color digital camera, and 5×, 10× and 20× objectives.
Rabbit anti mouse cd3 antibody
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-mouse CD3 antibody is a primary antibody that binds to the CD3 complex, which is expressed on the surface of T cells. This antibody can be used in various immunological techniques to detect and study mouse T cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555 conjugated anti rabbit igg, by Beyotime (2 mentions)
Lsm image examiner software, by Zeiss (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axiocam color digital camera, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Bz 9000, by Keyence (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Anti fluorescence quenching agent, by Wuhan Servicebio Technology (1 mentions)
Goat anti rabbit af488, by Abcam (1 mentions)
7 aad, by BD (1 mentions)
Egm 2 medium, by Lonza (1 mentions)
Goat anti rat hrp, by Jackson ImmunoResearch (1 mentions)
Peroxidase block, by Abcam (1 mentions)
Cathe m, by Biocare Medical (1 mentions)
Expose rabbit specific hrp dab detection kit, by Abcam (1 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
1x71 microscope, by Olympus (1 mentions)
Rabbit anti mouse antibody, by Abcam (1 mentions)
8 protocols using rabbit anti mouse cd3 antibody
1
Immunohistochemical Analysis of Splenic Tissue
2
Immunofluorescent Staining of FFPE Tissues
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Formalin-fixed paraffin-embedded tissues of 5-µm section were used for immunofluorescent staining. The following primary antibodies were used for the analysis: rabbit-anti mouse CD3 antibody from Abcam (Cambridge, UK), rat- anti-mouse B220 antibody from BD pharmngen (CA, USA). The following were used for the secondary antibodies: alexa Flour 647 donkey anti-rat from abcam, Alexa flour goat anti-rabbit from Abcam. The following primary antibodies were used as an isotype: rabbit IgG polyclonal isotype for CD3 (Abcam), purified rat IgG2a kappa Isotype control for B220 (Thermo Fisher Scientific K.K., Tokyo, Japan). DAPI was purchased from Vector laboratory (Burlingame, CA, USA). Images were acquired by a fluorescence microscope (BZ 9000, Keyence, Osaka, Japan).
3
Immunostaining of Vascular Cells
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Rabbit anti-mouse GFP antibody, Rabbit anti-mouse CD3 antibody and goat anti-rabbit AF488 were purchased from abcam; rat anti-mouse CD31, CD144 and MHC-II molecule monoclonal antibody were purchased from eBioscience; goat anti-rat or anti-rabbit Cy3, anti-fluorescence quenching agent and rabbit anti-mouse CD31 polyclonal antibody were purchased from Servicebio; goat anti-rat AF488 was purchased from CST; EGM-2 medium was purchased from Lonza; Fibronectin was purchased from EMD Millipore; Anti-Mouse-CD31-V450, anti-Mouse-VEGFR2-PE and anti-Mouse-CD45-PerCP Cy7 were bought from BD; 7-AAD was purchased from Nanjing KGI Biotech Co., Ltd.; mouse endothelial cell growth factor (VEGF) was bought from Proteintech; Accutase-Enzyme Cell Detachment Medium was purchased from Thermo Fisher Company.
4
Immunohistochemical Staining of Tumor Sections
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MC38 tumors were embedded in optimal cutting temperature (OCT) compound, snap-frozen on dry ice, and stored at -80°C until staining. Serial 5 μm tumor sections were placed onto glass slides, dried overnight at room temperature (RT), and then stained or stored at -80°C until use. For immunohistochemical staining, air-dried slides were fixed with acetone at RT, treated with peroxidase block (Abcam) to quench endogenous peroxidase, and then further blocked with a 10% goat serum and 5% BSA solution. Sections were stained with rabbit anti-mouse CD3 antibody (Abcam SP7) at a 1:400 dilution or rat anti-mouse PD-L1 (eBioscience MIH5) at a 1:100 dilution in blocking buffer. CD3+ T cells were detected using the EXPOSE rabbit-specific HRP-DAB detection kit (Abcam ab80437). PD-L1+ cells were detected using goat anti-rat-HRP (1:1000; Jackson ImmunoResearch 112-036-071) and detected with the HRP-DAB detection kit. Slides were co-stained with pre-made hematoxylin (Biocare CATHE-M) to counterstain cell nuclei. A similar protocol was applied for staining of PD-L1.
5
Immunohistochemical Staining of Murine Uterus
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Uterus were collected from mice after cardiac perfusion with PBS and fixed in 4% formaldehyde solution immediately and made into paraffin sections. Paraffin sections were stained with hematoxylin and eosin (HE). Paraffin sections of uterus were rehydrated and boiled in EDTA buffer (pH 6.0) for 20 min to induce antigen retrieval. After washing, tissue sections were blocked with 5% goat serum for 30 min at 37°C, followed by staining with rabbit anti-mouse CD3 antibody (Abcam, polyclone, 1:100) and armenian hamster anti-mouse γδT antibody (Santa Cruz Biotechnology, clone: UC7-13D5, 1:100) at 4°C overnight. Sections were washed four times for 5 min each time and incubated with Alexa Fluor 555-conjugated anti- rabbit IgG (Beyotime, 1:1000) plus DyLight 488-conjugated anti-Syrian hamster IgG (Abcam, 1:1000) for 30 min at 37°C in the dark. After a final washing, cover slips were mounted onto slides with fluoroshield mounting medium with DAPI (4, 6-diamidino-2-phenylindole, Abcam). Images were captured with Olympus microscope BX53 and processed with LSM Image Examiner software (Zeiss).
6
Immunofluorescent Staining of Lung Tissues
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Paraffin sections of lung tissues from wild type (WT) and infected mice were rehydrated and boiled in Sodium citrate buffer (pH 6.0) for 30 min to induce antigen retrieval. After washing, tissue sections were blocked with 10% goat serum, followed by staining with rabbit anti-mouse CD3 antibody (Abcam) and hamster anti-mouse γδT antibody (Santa Cruz Biotechnology) at 4°C overnight. Sections were washed and incubated with Alexa Fluor 555–conjugated anti-rabbit IgG plus Alexa Fluor 488–conjugated anti-hamster IgG (Beyotime, Shanghai, China) for 30 min at 37°C in the dark. After a final washing, cover slips were mounted onto slides with fluoroshield mounting medium with DAPI (Abcam). Images were captured with Olympus microscope BX53 and processed with LSM Image Examiner software (Zeiss).
7
Immunohistochemistry of Liver and Kidney Tissues
8
Multimarker Immunohistochemistry in Kidney
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Immunohistochemical detection of macrophages, lymphocytes and myofibroblasts (using α-SMA) was performed on paraffin-embedded kidney sections. Briefly, after dewaxing and rehydration, a microwave pre-treatment in citrate buffer (pH 6.2) was performed to unmask antigens present in the renal tissue. Tissue sections were then incubated for 1 h with primary antibodies: anti-macrophages (rat anti-mouse F4/80 antibody, Abcam, UK), anti-lymphocytes (rabbit anti-mouse CD3 antibody, Abcam, UK) and anti-α-SMA (rabbit anti-mouse antibody, Abcam, UK). After rinsing in PBS, slides were exposed for 30 min to the appropriate secondary antibody. Kidney sections were finally incubated with ABC complex (Vector Laboratories, UK) for 30 min and bound peroxidase activity was detected with the DAB kit (DAKO, Belgium). Counterstaining was performed with hemalun and Luxol fast blue.
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