Rabbit anti gpr81

Cells were plated on chamber slides and maintained overnight at 37°C in a mixture of 5% CO₂ and 95% air in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were fixed in acetone and blocked with 4% fish gelatin for 20 min. Rabbit anti-GPR81 (Abnova, Walnut, CA) was used at 1:120 dilution and incubated overnight at 4°C. Negative controls were done using isotype control antibodies (Jackson ImmunoResearch, West Grove, PA). Following washes, the appropriate fluorophore-conjugated secondary antibody was added (Jackson Immunoresearch, West Grove, PA), nuclei stained with Hoescht (1μg/ml), and slides covered using VECTASHIELD mounting medium (Vector laboratories, Burlingame, CA). Sections were examined on a Zeiss Axioplan2 microscope and images captured with a Hamamatsu ORCA-ER camera with Image-Pro Plus software (Media Cybernetics, Rockville, MD) and analyzed using Simple PCI software (Hamamatsu Corporation, Sewickley, PA).

Tissues were either fixed in 4% formalin then embedded in paraffin or snap frozen in liquid nitrogen and embedded in ornithine carbamyl transferase medium and sectioned. Paraffin embedded sections were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed with diva decloaker (Biocare Medical, Concord, CA) in a steamer for 20 minutes. Endogenous peroxidase was blocked with 4% H₂O₂ and protein-blocked with 4% fish gelatin. Frozen sections were fixed in acetone, briefly air dried, and blocked with 4% fish gelatin for 30 minutes. Primary antibodies were incubated overnight at 4°C and included: rabbit anti-GPR81 (Abnova, Walnut, CA), 1:50 dilution; rabbit anti-MCT1 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:50 dilution; rabbit anti-PGC1α (Novus Biologicals, Littleton, CO), 1:50 dilution; anti-Ki67 (Thermo Fischer Scientific, Waltham, MA). Negative controls were done using isotype control antibodies (Jackson ImmunoResearch, West Grove, PA). Following washes, VECTASTAIN^® ABC systems (Vector laboratories, Burlingame, CA) was added to paraffin sections per manufacturer protocol, developed with 3,3-diaminobenzidine substrate, counterstained with hematoxylin and mounted with water soluble mounting media. Frozen sections were developed with fluorophore-conjugated secondary antibody, as described above.