The primary antibodies used were: rabbit anti-CKIIα (2656), rabbit anti-BAF60c (62265), and rabbit anti-phospho-histone H3 (Ser10; D2C8) conjugated to Alexa Fluor 647 and were obtained from Cell Signaling Technologies. The rabbit anti-Brg1 (G-7), rabbit anti-BAF155 antibody (H-76), rabbit anti-lamin β1 (H-90), and rabbit anti-pol II (N-20) were purchased from Santa Cruz Biotechnology. The rabbit anti-PI3K p85 antibody, N-SH2 domain (ABS233), was obtained from Millipore Corp. The rabbit anti-Ini1 (BAF47) antisera was previously described [29 (link)]. The rabbit anti-SMARCE1 (BAF57, A13353) and rabbit anti-SMARC1 (BAF60a, A6310) were obtained from Abclonal Technology. The secondary anti-mouse and anti-rabbit horseradish peroxidase-conjugated antibodies and the Alexa Fluor 488- and 555-conjugated antibodies were from Thermo Fisher Scientific.
Alexa fluor 488 and 555 conjugated antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488- and 555-conjugated antibodies are fluorescently labeled secondary antibodies used for detection and visualization in various immunoassays and imaging techniques. The Alexa Fluor dyes provide bright, photostable fluorescence signals that can be detected with standard fluorescence instrumentation.
Automatically generated - may contain errors
2 protocols using alexa fluor 488 and 555 conjugated antibodies
1
Chromatin Remodeling Complex Antibody Panel
2
Immunohistochemistry and Whole-Mount Staining
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For IHC, cultures were cut with a ϕ6 mm trephine blade (Kai Industries Co., Ltd., Gifu, Japan). Ten μm thick cryosections were fixed with ice-cold 4% paraformaldehyde (PFA) in PBS for 5 min. Sections were treated with PBS containing 0.1% Triton-X and 10% normal donkey serum to block non-specific staining. For WMS, cultures were fixed with ice-cold 4% PFA for 10 min, cut by ϕ6 mm trephine, and permeabilized with 0.5% Triton-X in PBS at RT for 20 min. Mouse monoclonal antibodies for KRT3 (sc-80000, AE5, Santa Cruz Biotechnology, Inc., Santa Cruz, Dallas, TX), KRT15 (ab-1385, LHK15, Abcam, Cambridge, MA), p63 (sc-8431, 4A4, Santa Cruz), PMEL (sc-52704, NK1/betab, Santa Cruz), chicken anti-KRT15 antibody (PCK-153P, Covance, Princeton, NJ), and rabbit antibodies for KRT12 (sc-25772, Santa Cruz), PAX6 (RPB-278P, Covance), MELANA (MAB1656, Abnova, Taipei, Taiwan), TJP1(402200, Thermo Fisher) were used as primary antibodies. Mouse IgG1 (MOPC21, M5284, Sigma-Aldrich) and mouse IgG2a (UPC-10, M5409, Sigma-Aldrich) were used as isotype control. Alexa fluor 488 and 555 conjugated antibodies (Thermo Fisher) were used as secondary antibodies. IHC sections were treated with antibodies for 1 hour, whereas WMS samples were treated for 2 hours. Nuclei were counterstained with DAPI. Images were obtained by fluorescent microscope.
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