Donkey anti rabbit secondary antibody

Erythrocyte LPCAT activity was determined by measuring the formation of [¹⁴C] PC from lysoPC and [¹⁴C] acyl-CoA26 (link). All reactions were performed in 100 mM Tris-HCl, pH 7.4 containing 1 μM CaCl2, 0.015% Tween-20, 200 μM lysoPC (Avanti Polar Lipids), and 20 μM [1-¹⁴C] oleoyl-CoA (0.01 μCi) (Perkin Elmer) at 37 °C with isolated erythrocyte membrane protein in a total volume of 200 μl. The reaction was terminated by adding 800 μl of chloroform/methanol (2:1, vol/vol) to the incubation mixture. Then lipids were extracted and then resolved by Thin-layer chromatograph (TLC) silica plates with chloroform/methanol/acetic acid/0.9% NaCl (100:50:16:5, vol/vol). TLC plates were then exposed to phosphor-imaging screening (Bio-Rad) and scanned for radioactive signals as indications of the formation of PC. A part of membrane protein was used to detect LPCAT1 protein expression by Western blot. Approximately 20 μg membrane protein was run on 10% SDS-PAGE gels. After protein was transferred to PVDF membrane, the membrane was blocked with 5% nonfat milk and incubated with anti-LPCAT1 antibody (ab94903, Abcam) and Donkey anti-Rabbit secondary antibody (Santa Cruz Biotechnology Inc.), respectively.

Collagen1-α immunofluorescence was performed in paraffin-embedded sections of mouse lung. Before immunostaining, antigen retrieval was performed by heating slides in pH 6.0 citrate buffer at 100°C for 20 min in a microwave oven at 500 W using antigen retrieval solution (10 mM Tris and 1 mM EDTA, pH 9.0). Non-specific antibody binding was blocked for 20 min by incubation with 0.05% w/v BSA in PBS. Slices were stained using the anti-collagen1-α antibody (Novus, Littleton, CO, USA, 1:100 dilution) at 4°C overnight followed by staining with donkey-anti-rabbit secondary antibody (Santa Cruz, CA, USA, 1:200 dilution) at 37°C for 2 h.