Rabbit anti fasn polyclonal antibodies

Proteins were extracted from lung tissues or cells in lysis buffer (Thermo Fisher Scientific) using protease and phosphatase inhibitor cocktail (Roche Diagnostics), followed by centrifugation. Western blotting was performed as described previously^{36 (link)}. For each experiment, equal quantities of total protein were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blots were subsequently blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: mouse anti-6X His tag monoclonal antibody (1:1000; Abcam), rabbit anti-FASN polyclonal antibodies (1:500; Abcam, Cambridge), rabbit anti-caspase-3 (1:500; Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1:500; Cell Signaling Technology), rabbit anti-PINK1 (1:500; Proteintech) and anti-β-actin monoclonal antibody (1:5000; Sigma-Aldrich). After washing several times with TBS containing Tween (TBST), the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody (GenDEPOT). The membranes were analyzed by chemiluminescence (Thermo Fisher Scientific and Bio-Rad, Berkeley, CA, USA) using a ChemiDoc™ Touch Imaging System (Bio-Rad).

Lung tissues were dehydrated and embedded in paraffin. For histological examination, sections 4 µm thick and bronchoalveolar lavage fluid (BALF) cells on slides were treated with 1.4% H₂O₂–methanol for 30 min to block endogenous peroxidase. Then, nonspecific binding was blocked with 1.5% normal serum, and slides were incubated with rabbit anti-FASN polyclonal antibodies (1:200; Abcam, Cambridge, UK), mouse anti-6X His tag monoclonal antibody (1:200; Abcam). The next day, the sections were incubated with ABC kit reagents (Vector Laboratories, Burlingame, CA, USA). The color reaction was developed by staining with a liquid 3,3′-diaminobenzidine positive-substrate kit (Golden Bridge International, Inc., Mukilteo, WA, USA). After immunohistochemical staining, the slides were counterstained with Harris’s hematoxylin for 1 min.