Immunocytochemistry for autophagy

To determine if cells were undergoing autophagy, expression of MAP1LC3A, one of the proteins involved in the formation of autophagosomes, was determined [36,37]. The cells were grown on chamber slides (Lab-tek; Nalge Nunc International, Naperville, IL, USA) at a density of 10×10⁴ cells/slide. At 24 h prior to genistein treatment the media were changed to DMEM/F-12 (Sigma) phenol red free with charcoal/dextran treated FBS (Hyclone) for both cell types. At 72 h, genistein-treated and control cells were used for immunostaining. The cells were washed with 1X automation buffer (Biomeda Corporation, Foster City, CA, USA), then fixed in 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.2% triton X-100 (Sigma) and quenched with 0.3% hydrogen peroxide (Sigma), for 20 min each. Both cell types were blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in 5% BSA (Sigma) and an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) for 30 min. The cells were incubated overnight in the primary antibody, Authopagy APG8a (MAP1LC3A) rabbit polyclonal antibody (AP1801a, 1:200, Abgent, San Diego, CA, USA), in 5% BSA (Sigma), followed by a 30 min incubation with the secondary antibody, a biotinylated donkey anti-rabbit (711-065-152, 1:500, Jackson) that was subsequently labeled with the Vectastain Standard Elite ABC kit (Vector) for 30 min. Cells were incubated with 3,3’-diaminobenzidine (DAB) chromogen (DAKO, Carpinteria, CA, USA) for 6 min and counterstained for 30 sec using Mayer's hematoxylin (Poly Scientific, Bayshore, NY, USA). During the staining procedure, nonimmune rabbit serum (Jackson), at the same concentration as the primary antibody, served as the negative control. Lastly, the slides were scanned and images were captured at 40X using an [38] Aperio ScanScopeXT and the Aperio ImageScope software (v11.0.2.716; Aperio Technologies, Vista, CA, USA).