Fasn antibody

For each paraffin-embedded specimen, 5 µm thick sections were baked, deparaffinized, and hydrated as described previously^{38 (link)}. Specimen washing and antigen retrieval were done with sodium citrate solution. Sections were incubated overnight at 4 °C with primary antibodies : rabbit polyclonal FASN antibody (1:200; abcam), mouse monoclonal SPC antibody (1:100; Santa Cruz Biotechnology) and rabbit polyclonal PINK1 antibody (1:100, Proteintech). Antibodies were diluted in 1 × phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) and 0.3% Triton X100. Secondary antibodies, Alexa Fluor 568 goat anti-rabbit immunoglobulin G (IgG) (H + L) (abcam) and Alexa Fluor 488 goat anti-mouse IgG (abcam) were used at 1:200 dilution. These were incubated for 2 h at room temperature. Nuclei were counterstained with DAPI stain solution. As a negative control, and to ensure specificity of staining, the primary antibodies were omitted from the staining procedure on some samples. Tissue were imaged using an Confocal Microscope (Leica, Germany). Images were processed using ImageJ 1.44 (WayneRasband, NIH, USA).

The cells were washed twice with ice-cold PBS and directly lysed in 200 μl of cell lysis buffer. The lysates were boiled, centrifuged at 10000 rpm and then loaded onto a 6% SDS–PAGE gel. The samples were electrophoresed for 2 h and transferred onto transfer membranes. After being blocked with 5% BSA in PBS–Tween 20 for 1 h at room temperature, the membranes were blotted with FASN antibody (Abcam, Cambridge, MA, USA) or α-Tubulin primary antibodies (Beyotime, Nantong, China) at 1:1000 dilutions. The membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibody at a 1:2000 dilution for 1 h at room temperature. After the membranes were washed with Tris-buffered saline–Tween 20, the blots were incubated with enhanced chemiluminescence (ECL) stable peroxide solution (Beyotime, Nantong, China). All blots were visualized using a FluoroChem MI imaging system (AlphaInnotech, Santa Clara, CA, USA) at room temperature.

The expression of FASN was detected by immunohistochemistry (IHC) using a commercial FASN antibody (dilution 1 : 200; Abcam, Cambridge, UK). The IHC assay was conducted according to the manufacturer's instructions with all the staining reagents. The evaluation of IHC staining was performed by two independent pathologists without knowing the patients' clinical information using a modified histological score method based on both the percentage of positively stained cells and the intensity of staining. Under 40× visual field magnification, the intensity score (I) of staining was classified into four grades (ranks 0 to 3), while the extent score (E) of stained cells was classified into five grades (ranks 0 to 4). The IE score was constructed as follows: intensity score (I) × extent score (E), with a maximum score of 12. If the interobserver variability exceeded 15%, the sample sides would be rescored to reach a consensus for each patient's slide. We arbitrarily defined IE score (0-5) as low expression and IE score (6-12) as high expression.