GC tumors from mice were immunostained for Ki-67 with two-step immunohistochemical staining using streptavidin–peroxidase (SP) and diaminobenzidine (DAB). Fresh gastric tumor biopsies collected from nude mice were fixed immediatelsy with 4% paraformaldehyde (Sigma-Aldrich, Irvine, Ayrshire, UK) for 30 min at 30°C. The biopsies were then embedded in paraffin, sectioned (4 μm thick) onto slides, and rehydrated in xylene. Antigen retrieval was performed in 20 mmol/L sodium citrate-repairing solution (pH 6.0) at 95°C for 15 min. The tissue sections were then incubated in 3% H2O2 solution for 10 min to block endogenous peroxidase activity and incubated with 1:50 diluted Ki-67 antibody (Boster Biological Technology, Ltd., Wuhan, P.R. China) at 4°C overnight. PBS solution as a substitute for primary antibody was used as the negative control. After rinsing three times with PBS, the tissue sections were further incubated with the appropriate secondary antibodies at 37°C for 30 min followed by exposure to SP for another 30 min. Subsequently, the tissue section was stained with DAB (Tiangen Biotechnology, Beijing, P.R. China), counterstained with hematoxylin, dehydrated, and sealed.
Ki 67 antibody
Manufactured by Boster Bio
Sourced in United States
The Ki-67 antibody is a laboratory reagent used for the detection and quantification of the Ki-67 protein, a cellular marker for proliferation. The Ki-67 protein is expressed during active phases of the cell cycle, making it a useful tool for assessing cell growth and division in various biological and clinical applications.
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2 protocols using ki 67 antibody
1
Immunohistochemical Analysis of Ki-67 in GC Tumors
2
Total Protein Extraction and Western Blot Analysis
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Total protein was extracted by Total Protein Extraction Kit (ProMab, USA). The total protein concentration was measured using the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, USA). An aliquot comprising 20 μg of total protein was used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane after blocking with defatted milk at 37°C for 2 hours and incubated at 4°C overnight with anti-PCNA antibody (2586, Cell Signaling Technology, USA) and ki67 antibody (BM438, Boster, China); antibodies for proteins involved in EMT used the same as IHC method, goat anti-mouse and rabbit (ab205719, ab205718) as second antibody. Immunoreactive proteins were detected using enhanced chemiluminescence (NCI4106, PIERCE, USA). Band was analyzed by Image-Pro Plus 6.0. Each experiment was performed in triplicate.
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