SiHa and CaSki cells were grown on glass coverslips (1×105 cells per well) in 24-well plates, fixed in 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.) for 15 min at room temperature and permeabilized with 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 30 min at room temperature. After blocking with 1% BSA (Sangon Biotech Co., Ltd.) for 15 min at room temperature, the coverslips were incubated with anti-ki67 (dilution, 1:200; cat no. A11390, ABclonal Biotech Co., Ltd.) or anti-p65 antibodies (dilution, 1:200; cat no. A19653, ABclonal Biotech Co., Ltd.) at 4°C overnight, followed by incubation with the Alexa Fluor™ 555-conjugated goat anti-rabbit (dilution, 1:200; cat no. A27039, Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Cell nuclei were stained with DAPI (Shanghai Aladdin Biochemical Technology Co., Ltd.) at room temperature for 5 min and observed under a fluorescence microscope. Total p65 and nuclear p65 levels were quantified. Relative p65 nuclear localization was expressed as the ratio of the fluorescence density of nuclear p65 staining to the fluorescence density of total p65 cellular staining.
Anti ki67
Manufactured by ABclonal
Sourced in China
Anti-ki67 is a laboratory reagent used for the detection of Ki-67, a protein associated with cellular proliferation. It is commonly used in immunohistochemistry and flow cytometry applications to identify actively dividing cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Sinopharm (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
A2094, by ABclonal (1 mentions)
Fluorescent labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Haematoxylin, by Solarbio (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti mettl3, by Abcam (1 mentions)
Anti fto, by Abcam (1 mentions)
Anti map1lc3 b, by ABclonal (1 mentions)
Goat anti rabbit igg fitc, by Abcam (1 mentions)
Anti ythdf1, by ABclonal (1 mentions)
Anti alkbh5, by Abcam (1 mentions)
Anti mettl14, by Abcam (1 mentions)
Anti bcl 2, by ABclonal (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
6 protocols using anti ki67
1
Immunofluorescence Assay for Cellular Localization
2
Ki-67 and TPRG1L Expression in Xenograft Tumors
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Xenograft tumors were fixated in 4% polyoxymethylene for 3 days, embedded in paraffin, and then cut to 5 mm thickness. After dewaxing, rehydration, and antigen retrieval, slides were incubated with anti-Ki-67 (A2094, Abclonal) and anti-TPRG1L (A15949, Abclonal).
3
Ki-67 Immunofluorescence Staining of Tumor Tissues
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Frozen sections (6 μm) of tumor tissues were fixed using acetone at −20 °C for 10 min. Cell membranes were permeated in PBS containing 0.1% Triton X-100 at room temperature for 8 min. The slices were blocked in 3% BSA/PBS at room temperature for 1 h and then incubated with the primary antibody (anti‐Ki‐67, Abclonal, Wuhan, China, 1:100) overnight at 4 °C. Subsequently, the slides were incubated with a fluorescent-labeled secondary antibody (Invitrogen, Carlsbad, California, USA, 1:2000) for 1 h at room temperature. Next, the nucleus was stained by DAPI (0.5 mg/mL) for 15 min at 37 °C. Images were photographed using a spectral laser scanning confocal microscope (Olympus, Japan) after the slices were sealed by antifade solution.
4
Immunohistochemical Analysis of Xenograft Tumors
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Paraffin-embedded sections of xenograft tumours from nude mice (5 μm) were dewaxed in xylene and rehydrated with distilled water in an ethanol gradient. These sections were then incubated with 3% H2O2 at room temperature for a period of 15 min, blocked with 1% bovine serum albumin, followed by overnight incubation at a temperature of 4 °C with anti-LAGE3 (1:100 dilution; Novus Biologicals, Littleton, CO, USA) or anti-ki67 (ABclonal, Wuhan, China). The subsequent incubation was conducted with horseradish peroxidase-conjugated secondary antibody (1:100 dilution; Thermo Fisher, USA) for a period of one hour at 37 °C. Finally, sections were re-stained with hematoxylin for 3 min after development with 100 μL of 3,3′-diaminobenzidine reagent.
5
Immunohistochemistry for SFRS9 and Ki67
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IHC staining for SFRS9 and Ki67 was performed. Tumor tissue sections were obtained from paraffin blocks and rehydrated, and then incubated overnight with the primary antibodies including anti-SFRS9 (1:100, ABclonal Technology, Wuhan, China) and anti-Ki67 (1:100, ABclonal Technology). The sections were then washed with PBS, and incubated with the appropriate HRP-conjugated secondary antibody (1:500 dilution, ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 1 h, and visualized with DAB. The haematoxylin (Solarbio) was used to counterstain the sections. Finally, the sealed slides were photographed under a microscope (BX53, Olympus).
6
Antibody Validation for m6A Pathway
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Anti-METTL3 (western blot [WB] 1:1,000; IHC 1:500; ab195352), anti-FTO (WB 1:1,000; IHC 1:500; ab124892), anti-METTL14 (WB 1:1,000; IHC 1:500; ab220030), anti-ALKBH5 (WB 1:1,000; IHC 1:500; ab174124), goat anti-rabbit immunoglobulin (Ig)G (WB 1:2000; ab6721) and goat anti-rabbit IgG-FITC (immunofluorescence [IF] 1:200, ab6717) antibodies were purchased from Abcam, USA. Anti-MAP1LC3-B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), Anti-MAP1LC3-B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), anti-Bcl-2 (WB 1:1,000; IHC 1:100; A), anti-YTHDF1(WB 1:1,000; IHC 1:100; A18126), anti-YTHDF2 (WB 1:1,000; IHC 1:100; A15616), anti-Ki67 (IHC 1:100; A11390), anti-GAPDH (WB 1:1,000; AC027) and anti-β-actin (WB 1:50,000; AC026) antibodies were purchased from Abclonal, China. Anti-m6A (Me-RIP 1:1,000; ABE572) antibody was purchased from Merck Millipore (Massachusetts, USA). Western blot analyses were performed by standard methods described previously.59 (link)
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