Plin5 antibody

Tissues were rapidly frozen in melting isopentane after being coated in OCT Tissue-Tek (Sakura Finetek, The Netherlands). Serial 10 μm sections were cut at −20 °C on to SuperFrost Ultra Plus glass slides. Slides were fixed in Bouin's solution with 0.1% Triton X-100 (Sigma–Aldrich) for 30 min. Sections were stained for lipid droplets with freshly made, filtered oil red O (Sigma–Aldrich) made in 60% triethylphosphate (Sigma–Aldrich). β-galactosidase expression was identified using commercial reagents (MIR 2600, Mirus Bio, Madison, WI, USA). Sections were blocked in PBS with 1% BSA for 1 h then incubated overnight with PLIN5 antibody (Cat. no. GP31, Progen Biotechnik, Germany, ∼0.02 mg/ml) while mitochondrial staining was performed using the mitochondrial antibody directed against the oxidative phosphorylation complexes I–V (Total OXPHOS Cat. no. ab110413, Abcam, Cambridge, UK). For negative controls, primary antibodies were substituted for concentration matched guinea pig serum for PLIN5 (Antibodies Australia), or OXPHOS cocktail pre-absorbed 4:1 overnight with mouse IgG₁ and IgG_2a (Dako, Denmark). Immunohistochemistry images were analyzed for colocalization with ImageJ (NIH) by using Manders' colocalization coefficient, M2, which provides a fraction of the colocalizing objects in the images. Electron microscopy was performed as described previously in Ref. [34] (link).