Bz x series fluorescence microscope

Immunohistochemical staining for PLIN2, a lipid droplet protein, was conducted as described previously (Rasineni et al., 2014 (link)). Paraffin-embedded liver sections were deparaffinized, rehydrated and treated with 10 mM sodium citrate buffer (pH 6) for 20 min for antigen retrieval. Liver sections were incubated with a PLIN2 antibody (Fitzgerald#10R-A117ax) overnight followed by the appropriate Alexa Fluor secondary antibody for 1 h. Vectashield mounting medium with DAPI was used for mounting sections. Images were visualized and captured using a Keyence BZ-X series fluorescence microscope.

Cultured cells were seeded as a monolayer on a chamber polystyrene vessel tissue culture treated glass slide (BD Biosciences). After approximately 70% confluence was reached, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, then washed 3 times with PBS. Cells were permeabilized using 0.2% Triton X-100 in PBS for 15 min and blocked with 5% goat serum in PBS with 0.1% Triton X-100 for 1 h. Cells were incubated with primary antibodies (1:200 diluted) in blocking buffer overnight at 4 °C. After washing three times with PBS-0.1% Triton X-100, fluorescence-conjugated secondary antibodies (1:500 diluted) were applied correspondingly. Isotype-specific IgG was used as the negative control. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Vectashield, Vector Laboratories, Burlingame, CA, USA). The analysis used a BZ-X Series fluorescence microscope (Keyence, Itasca, IL, USA).