Cat hematoxylin counterstain

The excised tissues were fixed in 4% paraformaldehyde (PFA) for 24 to 48 hours, transferred to 70% ethanol, processed, and embedded in paraffin. Sections were cut at 4 μm and mounted on slides for H&E and Periodic acid Schiff (PAS) staining. For IHC, sections were incubated with the primary antibodies for 60 minutes followed by incubation with immPRESS goat anti-rabbit or goat anti-mouse IgG polymer (catalogs MP-7602 and MP-7601, Vector BioLabs) for 30 minutes. The antigen-antibody complexes were detected using the ImmPACT DAB peroxidase (HRP) substrate (Vector BioLabs) and counterstained with CAT hematoxylin counterstain (Biocare) according to the manufacturer’s instructions. The following primary antibodies used for the IHC analysis included MAML2 TAD antibody (1:100, Bethyl IHC-00446), Ki-67 antibody (1:100, Dako, Agilent msxhu, 7240), cleaved caspase-3 antibody (1:400, Cell Signaling Technology, 9664), phospho-RB antibody (1:400, Cell Signaling Technology, 8516), Cdk4 antibody (1:300, Santa Cruz Biotechnology Inc., sc-166373), and P16 antibody (1:50, Santa Cruz Biotechnology Inc., sc-1661). TUNEL assays were performed using ApopTag Peroxidase In Situ Apoptosis Detection Kit (EMD Millipore, S7100) according to the manufacturer’s instructions. The stained tissue sections were scanned and digitized using Aperio Imagescope (Leica).

GFP immunohistochemistry was carried out by the Molecular Pathology Core at University of Florida. Briefly, 4-mm serial sections were deparaffinized and treated with citra (BioGenex, Fremont, CA) at 98°C for 30 min. Background Sniper (Biocare Medical, Walnut Creek, CA) was used to reduce unspecific background staining. Sections were incubated with rabbit anti-GFP (1:2000; Abcam, Cambridge, MA) for 60 min. The stain was visualized using MACH 2 Gt × rabbit HRP polymer (Biocare Medical), DAB chromogen (Biocare Medical), and CAT hematoxylin counterstain (Biocare Medical).

Slides were prepared as above and treated by Citra Steam (Biogenex, Fremont, CA, USA) for 30 min and 2.5% normal goat serum (Vector labs) for 20 min. The slides were incubated with primary antibody (Table S1) for 60 min. Secondary antibody (Table S1) was applied after washing. Stain was visualized using the DAB (3,3′-Diaminobenzidine) chromagen (Vector Laboratories, Burlingame, CA, USA) and CAT hematoxylin counterstain (Biocare Medical, Walnut Creek, CA, USA). Slides were scanned (Aperio Scanscope CS, Leica Biosystems, Wetzlar, Germany) and data were analyzed by ImageScope software (Leica Biosystems, Wetzlar, Germany).