Anti rabbit igg ap

Proteins were analyzed by SDS-PAGE using the NuPAGE system (ThermoFisher Scientific). Separated proteins were either identified by mass-spectrometric analysis or electrotransferred onto a nitrocellulose membrane by tank blotting with transfer buffer (ThermoFisher Scientific) according to the manufacturer’s instructions. The membranes were blocked with Universal Blot Buffer plus (Euroimmun) for 15 min and incubated with human serum or the polyclonal antibody against ITPR1 in Universal Blot Buffer plus for 3 h, followed by three washing steps with Universal Blot Buffer (Euroimmun), a second incubation for 30 min with anti-rabbit IgG-AP (Sigma-Aldrich), three washing steps, and staining with NBT/BCIP substrate (Euroimmun).

Protein was extracted from cells as previously described (51 (link)). For Western blotting analysis, 30 to 40 μg of protein was resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with specific primary antibodies in blocking buffer (Tris-buffered saline, 0.1% Tween-20, and 5% milk or bovine serum albumin), and detected with alkaline phosphatase (AP)–conjugated secondary antibody. The antibodies used were anti-NPRC (1:2000; Novus Biologicals; #NBP1-31365), anti-PPARγ (1:2000; Cell Signaling Technology; #2435), anti-FABP4 (1:1000; Cell Signaling Technology; #2120), anti-ADIPOQ (1:1000; Cell Signaling Technology; #2789), anti-GAPDH (1:2000; Protein Tech; #10494-1-AP), and anti-rabbit IgG-AP (1:20,000; Sigma; #A3687).

For analysis of cell proliferation, phospho histone 3 (pH3) staining was performed in unilaterally injected embryos at stage 23 that were fixed with MEMFAT for 2 hours at room temperature. First, embryos were washed in 1x PBS for 5 x 10 minutes and then blocked for 1 hour in 1x PBS / 10% Horse Serum before being treated with a rabbit-anti-pH3 antibody (1:100; Millipore, Temecula, CA, USA), diluted in 1x PBS / 10% Horse Serum and incubated overnight at 4°C. The next day, embryos were washed in a solution of 1x PBS / 0.1% Tween-20 for 2 hours, whereby the medium was changed 4 times. For blocking, embryos were incubated in 1x PBST / 20% Horse Serum for 1 hour. Incubation with the secondary antibody was performed for 5 to 6 hours with 1x PBST / 20% Horse Serum / anti-rabbit-IgG-AP (1:1000; Sigma-Aldrich, Munich, Germany). Overnight, embryos were kept in 1x PBS / 0.1% Tween at 4°C. On the last day, embryos were transferred into a 24-well plate after several washing steps with 1x PBS / 0.1% Tween and then incubated with AP buffer for 10 minutes. Staining was performed with 500 μl of BM purple, and fixation, with MEMFAT. 30% H₂O₂ was used to bleach the. Then, pH3-positive cells were counted on both sides of the eye area.