Bx40 epifluorescence microscope

BAT samples were fixed in formalin (freshly prepared in 1.27 M formaldehyde, 0.1 M phosphate-buffered saline, pH 7.2) and embedded in Paraplast Plus (Sigma-Aldrich, USA) for non-serial 5-µm thick sections. These sections were placed onto glass slides for hematoxylin/eosin staining and digital images were acquired randomly (TIFF format, 36-bit color, 1,360×1,024 pixels) using an Olympus DP71 camera and an Olympus BX40 epifluorescence microscope (Olympus, Japan). At least 10 photomicrographs per animal were randomly measured with the software Image-Pro Plus 5.0 (Media Cybernetics, USA). Photomicrographs were used for selection of fat droplets. The digital images of the droplets were analyzed and their areas calculated; the resulting data are shown in a histogram.

Pancreas, liver, and retroperitoneal fat samples (n = 5−6 rats from 5 to 6 litters per group) were removed and fixed in 4% paraformaldehyde for 24 h. Subsequently, the samples were dehydrated in an alcohol-increasing series of concentrations. After diaphanization in xylene, the samples were embedded in histological paraffin. Slices of 5-μm thickness were prepared for staining with hematoxylin and eosin (H&E). In the pancreas and fat slices, islets (40 per animal, 40 × magnification) and retroperitoneal fat (20 per animal, 20 × magnification) photomicrographs were randomly acquired using an Olympus DP71 camera coupled to an Olympus BX40 epifluorescence microscope (Olympus, Tokyo, Japan). ImageJ for Windows (Open Source) was used for analysis. Liver slices (30 fields per animal, 20 × magnification) were examined under a light microscope. These values were classified as previously described for the magnitude of steatosis (32 (link)). Thus, steatosis was graded as follows: 0 (none to 5% of hepatocytes affected), 1 (>5%−33% affected), 2 (>33%−66% affected), and 3 (>66% affected). The predominant distribution pattern of steatosis was graded as follows: 0 (zone 3), 1 (zone 1), 2 (azonal), or 3 (panacinar).

iPSC-derived cultures were grown on coverslips and fixed in 4% paraformaldehyde/4% sucrose (w/v) in phosphate-buffered saline (PBS) solution. Cells were permeabilized in PBS with 0.4% v/v Triton X-100 for 2 × 7 min at room temperature (RT), then blocked in PBS with 10% v/v goat serum for 2 h at RT. GAD2 mouse monoclonal primary antibody (Chemicon, MAB351), diluted in PBS with 5% v/v goat serum (1:250), was applied for 2 h at RT. Goat anti-mouse Alexa Fluor 488 secondary antibody (Life Technologies A21131), diluted in PBS with 5% v/v goat serum (1:1000), was applied for 1 h at RT. Coverslips were washed three times for 5 min with PBS after each antibody application. Coverslips were then incubated with DAPI (Thermo Fisher Scientific; 1:5000 in PBS) for 5 min before being washed in PBS and briefly in dH₂O, then mounted using Prolong Diamond Anti-Fade Mounting Solution (Thermo Fisher Scientific). Images were acquired on an Olympus BX40 Epifluorescence microscope using HCImage imaging software and processed using ImageJ.