The slides were blocked in mouse-on-mouse (MOM) Ig blocking reagent for 1 hour followed by 2.5% normal horse serum for 5 minutes. They were then probed with mouse anti-α-SMA monoclonal antibody (Sigma A2547) diluted at 1:1500 for 30 minutes. After additional blocking with MOM reagent, the tissue was stained with Vector VIP peroxidase substrate (SK-4605) for 20 seconds. Next, blocking was performed with 0.1% BSA in PBS for 1 hour. Biotinylated HABP (EMD Millipore Calbiochem) was added at 1:250 dilution in PBS and incubated overnight at 4 °C. The next day, the slides were washed in PBS and treated with Vector Elite ABC reagent (PK-7100) for 30 minutes. Next, 3,3′-diaminobenzidine (DAB) peroxidase (Vector ImmPACT) was added for 1 minute. Finally, slides were washed in PBS and stained for 1 minute with preheated methyl green (Vector H-3402) at 37 °C.
The slides were then dehydrated in ethanol and replaced in Citrasolv. Coverslips were mounted with Cytoseal-60 (Richard-Allan Scientific; San Diego, CA). Imaging acquisition and scanning was performed with an Aperio Versa light microscope (Leica; Wetzlar, Germany) with ScanScope software.
The slides were then dehydrated in ethanol and replaced in Citrasolv. Coverslips were mounted with Cytoseal-60 (Richard-Allan Scientific; San Diego, CA). Imaging acquisition and scanning was performed with an Aperio Versa light microscope (Leica; Wetzlar, Germany) with ScanScope software.