Axioimager d1 fluorescent microscope

Liver tissues were fixed in neutral buffered 10% formalin solution (Sigma-Aldrich, HT501128-4L) embedded in paraffin and cut into 5-μm sections. Liver sections were deparaffinized with Histo-Clear I solution (Electron Microscopy Sciences, 64110-01) and hydrated through decreasing concentration of alcohol solutions. For catalase (peroxisomal marker) and VDAC1/2 (mitochondrial marker) staining, sections were unmasked 20 minutes at 600 W in a microwave with citrate buffer, pH 6.0. Samples were then blocked for 10 min with 3% H₂O₂ followed by 30 min with 2.5% normal goat serum. Sections were further incubated overnight at 4°C with primary antibodies (1:100 dilution) (catalase ref: ab16731; VDAC1/2, ab154856, Abcam) followed by 30 minutes of incubation with goat anti-Rabbit IgG Alexa Fluor™ 647 (for catalase staining) or goat anti-Rabbit IgG Alexa Fluor™ 488 (for VDAC1/2 staining) (1:200 dilution) (Thermo Fisher Scientific). Samples were mounted with Fluoromount-G mounting medium with DAPI (Invitrogen). All images were captured using a Carl Zeiss Axioimager D1 fluorescent microscope and quantified for number and size of peroxisome and mitochondria using Image J software.