Alexa fluor 555 conjugated goat anti rabbit immunoglobulin g

Immunofluorescence assays were conducted as described previously,⁹ (link) with minor modifications: primary rabbit anti-Myo7a (Abcore, Ramona, CA, USA), rabbit anti-GFP (Life Technologies, La Jolla, CA, USA), and mouse anti-GFP antibody (Millipore, Billerica, MA, USA) were diluted to 1:500, 1:500, and 1:200 in blocking solution, respectively. Secondary Alexa Fluor 555-conjugated goat anti-rabbit immunoglobulin G (IgG), Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, La Jolla, CA, USA) were diluted to 1:1,000 in blocking solution, respectively. Nuclei were stained with 1 μg/mL DAPI solution diluted in methanol for 5 min at RT. The samples were washed with PBS to remove any residual antibodies and mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using a fluorescence microscope (Axio Imager A2; Carl Zeiss, Oberkochen, Germany).

Cells were fixed with 4% paraformaldehyde for 15 min at RT and washed three times with phosphate-buffered saline. Then, the cells were incubated with 5% horse serum albumin in phosphate-buffered saline containing 0.1% Trion X-100 at RT for 30 min to block the unspecific binding of antibodies. After the blocking solution had been washed out, cells were incubated overnight at 4°C with rabbit anti-PPAR γ (1:100, sc-7196; Santa Cruz). Then, the cells were washed and incubated for 2 h at RT with Alexa Fluor^® 555-conjugated goat anti-rabbit immunoglobulin G (1:1000, A-21434; Invitrogen) and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL, D9542; Sigma-Aldrich) for 10 min. Fluorescent images were captured with a fluorescence microscope (LSM 510 META; Carl Zeiss, Stuttgart, Germany).