After fixation, deparaffinization and rehydration, the slides were incubated with donkey serum to reduce the non-specific antibody bindings, and then incubated with rabbit anti-mouse CD31 monoclonal antibody and mouse anti-mouse alpha-smooth muscle actin (α-SMA) monoclonal antibody (Abcam, Cambridge, MA, USA). Then, the slides were incubated with donkey anti-rabbit IgG antibody conjugated with Alexfluro 555 and donkey anti-mouse IgG antibody conjugated with Alexfluro 488 (Invitrogen, Carlsbad, CA, USA). The slides were mounted with mounting solution with DAPI (Vector Laboratories, Burlingame, CA, USA). The slides were examined and photographed under a fluorescence microscope.
Rabbit anti mouse cd31 monoclonal antibody
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-mouse CD31 monoclonal antibody is a laboratory reagent used for the detection and localization of the CD31 protein in mouse tissues. CD31, also known as PECAM-1, is a cell adhesion molecule expressed on the surface of endothelial cells and is commonly used as a marker for these cells.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidine tetrahydrochloride, by Santa Cruz Biotechnology (1 mentions)
Pharmingen, by BD (1 mentions)
4 protocols using rabbit anti mouse cd31 monoclonal antibody
1
Immunofluorescent staining of blood vessel markers
2
Immunohistochemical Analysis of Angiogenesis
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After micro-CT analysis, specimens were decalcified with 10% ethylene diamine tetraacetic acid solution for 1 week, dehydrated through an ethanol series, cleared with xylene, and then embedded in paraffin. The specimens were cut into 10-μm-thick sections and stained with HE.
Expression of CD31 was detected using rabbit anti-mouse CD31 monoclonal antibody (Abcam, Cambridge, UK). This was followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit antibody (Invitrogen) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz, CA, USA). Five randomly selected fields from each tissue section (n = 3/group) were captured by a light microscope (Olympus). The number of CD31-positive blood vessels was calculated by Image-Pro Plus software (Media Cybernetics).
Expression of CD31 was detected using rabbit anti-mouse CD31 monoclonal antibody (Abcam, Cambridge, UK). This was followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit antibody (Invitrogen) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz, CA, USA). Five randomly selected fields from each tissue section (n = 3/group) were captured by a light microscope (Olympus). The number of CD31-positive blood vessels was calculated by Image-Pro Plus software (Media Cybernetics).
3
Immunohistochemistry of Mouse Tumor Xenografts
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Immunohistochemistry staining was performed using Biotin-Streptavidin HRP Detection Kit (Zhongshan Bio, Beijing, China), according to the manufacturer’s procedure.25 (link) In brief, tumor tissues obtained from HepG2, CAFs combined with HepG2, or sh-VEGF-CAFs combined with HepG2 nude mice xenografts were formalin fixed, paraffin embedded, and then cut into 5-µm sections. After antigen retrieval with autoclaving in citric acid, and inactivating endogenous peroxidase with 3% H2O2, the slides were incubated with the rabbit anti-mouse CD31 monoclonal antibody (1:100 dilution; Abcam) overnight at 4°C. Second antibody conjugated with biotin was applied for 1 hour at room temperature. Then the sections were developed in 3,3-diaminobenzidine and counterstained with hematoxylin.
4
Quantifying Tumor Angiogenesis via PCNA and CD31
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The expression of PCNA and CD31 were estimated by immunochemistry with PCNA and CD31 antibodies. Briefly, the frozen tumor tissues were fixed in acetone and washed twice with PBS. Following, the handled samples were stained with rabbit anti-mouse CD31 monoclonal antibody (1:50; Abcam, Cambridge, UK) and rabbit anti-rat PCNA polyclonal antibody (1:50; BD PharmingenTM; BD Biosciences, San Jose, CA, USA). After washing twice with PBS, samples were stained with secondary antibody combining with FITC or Rhodamine. Finally, we surveyed the cells possessing-positive property under microscope (×400) and counted automatically the number of capillaries in 5 randomly selected fields.
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