Redmap

All IHC were performed using Ventana Discovery XT automated IHC/ISH slide staining system. Slides were cut at 4–5um. Deparaffinization and antigen retrieval were performed using CC1 (Cell Conditioning I, Ventana Medical Systems, Cat # 950-124). All primary antibodies were incubated at 37°C for 1 hour. Universal Secondary Antibody (Ventana Medical Systems, Cat#760-4205) was incubated for 12 minutes followed by chromogenic detection kit DAB Map (Ventana Medical Systems, Cat # 760-124) or Red Map (Ventana Medical Systems, Cat # 760-123). Slides were counterstained with Hematoxylin, then dehydrated and cleared before cover slipping from Xylene.

All IHC were performed using Ventana Discovery XT automated IHC/ISH slide staining system. Slides were cut at 4–5um. Deparaffinization and antigen retrieval were performed using CC1 (Cell Conditioning I, Ventana Medical Systems, Cat # 950-124). All primary antibodies were incubated at 37°C for 1 hour. Universal Secondary Antibody (Ventana Medical Systems, Cat#760-4205) was incubated for 12 minutes followed by chromogenic detection kit DAB Map (Ventana Medical Systems, Cat # 760-124) or Red Map (Ventana Medical Systems, Cat # 760-123). Slides were counterstained with Hematoxylin, then dehydrated and cleared before cover slipping from Xylene.

Deidentified breast tumor specimens exhibiting multiple morphological features were selected from archival FFPE tissue blocks at the Biospecimen Resource Shared Services at Rutgers Cancer Institute of New Jersey (IRB Protocol 009601). After a board-certified pathologist evaluated the quality of the specimens, tissue blocks were sectioned serially at 4 μm thickness. Two sections (#1 and #2) were immunohistochemically stained with estrogen receptor and progesterone receptor antibody (not shown). Section #3 was double stained with transformation-related protein (P63) in brown [3,3′-diaminobenzidine (DAB)] and α-smooth muscle actin (SMA) in red (RedMap; Ventana Medical Systems). Section #5 was de-paraffinized, but not stained, and kept in phosphate-buffered saline at room temperature before AFM probing. Section #6 was stained with hematoxylin and eosin (H&E) and once again quality controlled by a pathologist.

Paraffin slides were cut at 4–5um. Deparaffinization and antigen retrieval were performed using CC1 (Cell Conditioning Solution, Ventana Medical Systems, Cat # 950-124). Anti-P63 (Ventana Medical Systems, Cat#790-4509, mouse monoclonal antibody) was applied and slides were incubated at 37°C for 1 hour. Universal Secondary antibody (Ventana Medical Systems, Cat#760-4205) was incubated for 12 minutes followed by chromogenic detection kit DABMap (Ventana Medical Systems, Cat# 760-124). Slides were well-rinsed and re-labeled with proper protocols. Anti-SMA antibody (Ventana Medical Systems, Cat#760-2833, mouse monoclonal antibody) was applied and slides are incubated at 37°C for 1 hour. Universal Secondary antibody (Ventana Medical Systems, Cat # 760-4205) was incubated for 12 minutes followed by chromogenic detection kit RedMap (Ventana Medical Systems, Cat #760-123). Slides were counterstained with Hematoxylin, then dehydrated and cleared before cover slipping from Xylene.