Antivaspin antibody

Tissue preparation, lysis, Western blotting and quantification were performed as previously described (Rak et al. 2015a,b) . For each sample, 30 μg of protein were reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad), and then transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20, and then incubated overnight at 4°C with antivaspin antibody (cat no. PA5-30989, ThermoFisher Scientific) diluted at 1:1,000. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated antibody (cat. no. 7074, Cell Signaling Technology) diluted at 1:1000. An anti-β-actin antibody (cat no. A5316, Sigma-Aldrich) was used as loading control. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (cat. no. K-12043 D20, Advansta Inc., Menlo Park, USA) and visualised using the Chemidoc TM XRS + System (Bio-Rad). All visible bands were quantified using a densitometer and ImageJ software (US National Institutes of Health).

To determine vaspin localisation in the ovary or WAT, immunohistochemistry was applied (Reverchon et al. 2014) . Sections (5 μm thick) were mounted onto APES-coated slides, deparaffinised in xylene, and then gradually rehydrated through a series of ethanol dilutions. The sections were immersed in 0.01 M citrate buffer and heated in a microwave oven for antigen retrieval. Endogenous peroxidase activity and nonspecific binding were blocked. Next, the sections were incubated overnight at 4°C with anti-vaspin antibody (cat no. PA5-30989, ThermoFisher Scientific) then washed in TBST. After, they were incubated with biotinylated goat anti-rabbit IgG (1:400; Vector Lab) followed by avidin-biotin-peroxidase complex (1:1:100; Strept ABC complex/HRP, DAKO/AS). The sections were dehydrated, mounted in DPX (Fluka, Chemie GmbH, Buchs) and then photographed using the Nikon Eclipse E200 microscope attached to the Coolpix 5400 digital camera (Nikon) with corresponding software.