For immunofluorescent staining, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After two washes with PBS, the cells were incubated with blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Primary antibodies were diluted in blocking buffer and then added to the cells for 2 h at room temperature. The following primary antibodies were used: Bestrophin, MITF, PMEL17, AMPA R2 and Tuj1 (Abcam), ZO-1 (Thermo Fisher Scientific), Math5 (Millipore) and Brn3a (Santa Cruz). The cells were then subjected to three 3-min washes with PBS and incubated with secondary antibodies Alexa Fluor 488 or Alexa Fluor 594 of goat anti-rabbit or goat anti-mouse IgG (Thermo Fisher Scientific) diluted in blocking buffer containing DAPI (Thermo Fisher Scientific) for 1 h at room temperature. After three 3-min washes with PBS, the cells were visualized using confocal microscopy (Zeiss LSM 700).
Bestrophin
Manufactured by Abcam
Sourced in United States
Bestrophin is a laboratory equipment product. It functions as a calcium-activated chloride channel.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Brn3a, by Santa Cruz Biotechnology (1 mentions)
Pmel17, by Abcam (1 mentions)
Math5, by Merck Group (1 mentions)
Rhodopsin, by Merck Group (1 mentions)
Goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fv1000d, by Olympus (1 mentions)
2 protocols using bestrophin
1
Immunofluorescence Staining Protocol for Cell Analysis
2
Immunofluorescence Characterization of hiPSC-RPE
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The methods for immunocytochemistry and immunohistochemistry of hiPSCs and hiPSC-RPE were described previously. 22 Antigens detected with primary antibodies (species, company, and dilution) were Pax6 (rabbit; Covance, Princeton, NJ, USA, 1/600), Mitf (mouse; Abcam, Cambridge, MA, USA, 1/ 1000), Bestrophin (mouse; Abcam, 1/500), RPE65 (rabbit; 1/ 1000), ZO-1 (rabbit; Zymed Thermo Fisher Scientific, Waltham, MA, USA, 1/100), rhodopsin (mouse; Sigma-Aldrich Corp., 1/ 1000), and human EMMPRIN (mouse; R&D, Minneapolis, MN, USA, 1/80). The bound primary antibodies were detected with secondary antibodies labeled with Alexa Fluor 488, goat antirabbit IgG (Invitrogen, 1/500), or goat anti-human IgG (Invitrogen, 1/500) or Alexa Fluor 546, goat anti-mouse IgG (Invitrogen, 1/1000), or goat anti-rabbit IgG (Invitrogen, 1/ 1000), and the nuclei were stained with 4 0 6-diamindino-2phenylindole (DAPI) (1 lg/mL; Molecular Probes, Thermo Fisher Scientific). The samples were imaged using a laser scanning confocal microscope (FV1000-D; Olympus, Tokyo, Japan).
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