Rabbit anti mct1

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.