LSCs were cultured in six-well plates and treated with SP or AKT inhibitor MK-2206 for 5 days. The samples were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 min followed by blocking using 5% goat serum for 30 min at room temperature. Incubation with the following antibodies was performed at 4°C overnight: anti-Ki67 (1:50, Cat#ab279653, Abcam) and anti-DeltaNp63 (1:50, Cat#ab203826, Abcam). Next, incubation with Alexa Fluor-conjugated secondary antibodies (1:500, Cat#A-21422/A-11008, Thermo Fisher Scientific) was carried out. The nuclei were stained using DAPI (Cat#FD9637, Fude Biological Technology). Finally, the cells were visualized using a laser scanning confocal microscope-LSM 880 with Airyscan (ZEISS, Oberkochen, Germany).
Ab203826
Manufactured by Abcam
Ab203826 is a primary antibody for use in research applications. This product is a rabbit monoclonal antibody that recognizes a specific target protein. The core function of this antibody is to serve as a detection reagent for the target of interest.
Automatically generated - may contain errors
2 protocols using ab203826
1
Immunofluorescence Analysis of LSC Proliferation
2
Characterization of Limbal Stem Cell Markers
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Using Western blot (WB), the expression of typical LSC markers ATP-binding cassette superfamily G member 2 (ABCG2), p63, anti-p40-deltaNp63, and vimentin was measured as described in the literature [4 (link), 11 (link), 12 (link)]. First, cellular proteins from cLSCs were extracted using RIPA buffer with protease inhibitors. This extract was kept on ice for 30 minutes, and the lysis mixture was centrifuged at 11,000 g and 4°C for 15 minutes. The supernatant was transferred to a new tube, and the protein concentration was determined using the bicinchoninic acid kit (BCA, SERVA).
For WB, 30 μg of protein was used, and the membranes were incubated with mouse anti-ABCG2 (Abcam, ab108312), mouse anti-p63 (Abcam, ab124762), anti-vimentin (Santa Cruz Biotechnology, sc-6260) and rabbit anti-deltaNp63 (Abcam, ab203826) at 1:1000 dilution. As a positive control for ABCG2, p63 and deltaNp63, a protein lysate from the HeLa cell line (Merck) was used, and in the case of vimentin, a protein lysate of human MSC from adipose tissue (hAd-MSC) was used as a positive control. Secondary anti-mouse (Merck) and anti-rabbit (Abcam) antibodies were used for detection by ECL (SERVA), and the results were visualized in the ChemiDocTM XRS + system (Bio-Rad).
For WB, 30 μg of protein was used, and the membranes were incubated with mouse anti-ABCG2 (Abcam, ab108312), mouse anti-p63 (Abcam, ab124762), anti-vimentin (Santa Cruz Biotechnology, sc-6260) and rabbit anti-deltaNp63 (Abcam, ab203826) at 1:1000 dilution. As a positive control for ABCG2, p63 and deltaNp63, a protein lysate from the HeLa cell line (Merck) was used, and in the case of vimentin, a protein lysate of human MSC from adipose tissue (hAd-MSC) was used as a positive control. Secondary anti-mouse (Merck) and anti-rabbit (Abcam) antibodies were used for detection by ECL (SERVA), and the results were visualized in the ChemiDocTM XRS + system (Bio-Rad).
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