Anti perilipin antibody

For immunohistochemical staining of M1 macrophages in epididymal adipose tissue, the tissues were harvested and fixed in alcoholic formaldehyde (4% formaldehyde in 95% ethanol). After dehydrating and embedding in paraffin, 5 µm sections were cut on a Leica microtome (RM2255, Leica Microsystems) and mounted on SuperFrost microscopy slides (Thermo Fisher). Sections were deparaffinized, rehydrated and boiled for 10 min in target retrieval solution (DAKO) to unmask antigens. Afterwards, autofluorescence was blocked by incubation in 0.3% Sudan black in 75% ethanol for 10 min. The slides were rinsed with PBS and then blocked for 1 h at RT in Blocking Buffer (Roth). Anti-iNOS antibody (#129372, Abcam, 1:200) and anti-Perilipin antibody (#9349, Cell signaling, 1:1000) were diluted in Blocking Buffer (BB) and slides were incubated in primary antibodies over night in a humid chamber at 4 °C. After 3 times 10-min washing, slides were incubated for 1 h at RT in secondary antibodies (Jackson Immunoresearch, Cy2-anti rabbit and Cy3 anti-mouse, both 1:500 in BB). After additional 3 times washing in PBS, slides were mounted using Prolong Gold Antifade Mountant (Thermo Fisher). Microscopy was performed on a Nikon A1 confocal microscope (Nikon) using a Plan Fluor 40× Oil objective. Image analysis was performed using NIS Elements Advanced Research software (Nikon).

For protein analysis, cells were harvested in SDS lysis buffer and sonicated. The determination of protein concentration was done using the Compat-Able Protein Assay Preparation Reagent Set (Thermo Scientific, #23215, Vienna, Austria) and the Pierce BCA Protein Assay Kit (Thermo Scientific, #23227, Vienna, Austria). Samples (20 µg total protein) were separated on a 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membranes were probed with anti-perilipin antibody (Cell Signaling, #9349, Frankfurt am Main, Germany), anti-PPARγ2 antibody (Cell Signaling, #2435, Frankfurt am Main, Germany), anti-FABP4 antibody (Cayman, #10004944, Tallinn, Estonia), anti-CD24 antibody (Abcam, #179821) and anti-GAPDH (Thermo Scientific, AM4300, Vienna, Austria). Goat anti-rabbit IgG-HRP (DAKO, Vienna, Austria) and goat anti-mouse IgG-HRP (Promega, Walldorf, Germany) served as secondary antibodies and signals were detected with a chemiluminescence detection system. Membranes were stained with Ponceau S for normalization to total protein. Densitometric analysis was done using ImageJ software.

2×10⁴ C2C12 cells were seeded in 96 well plates and stimulated with or without 250 ng/mL of BMP6 or BMP7. Following adipogenic differentiation described in Figure 1A, cells were fixed in 4% PFA for 30 mins at RT, washed and permeabilized with 0.5% Triton-X 100 for 20 mins at room temperature. After washing, cells were blocked in 2% normal donkey serum and subsequently stained with anti-Perilipin antibody (9349, Cell Signaling Technologies) used at 1:200 dilution overnight at 4°C. The following day cells were washed and stained with HCS CellMask Green, Hoechst 33342 and donkey anti-rabbit Alexa-Fluor 647 (Life Technologies). Images were acquired on a Leica SP5II laser scanning confocal microscope at 63× and 63× with 3× scan zoom with the pinhole set at 1 airy.