Axioimager microscope

5-µm tissue sections were mounted on polylysine-coated glass slides (Thermo Fisher Scientific). Sections were washed in xylene and rehydrated using decreasing ethanol concentrations and finally in PBS. Endogenous peroxidase activity was quenched by immersing the slides in 3% hydrogen peroxide for 30 min. Antigen retrieval was performed using a programmable pressure cooker with target retrieval solution, pH 6.0 (Dako). Nonspecific reactivity in the tissues was blocked by incubation in 10% goat serum in PBS before incubating with the primary antibody at room temperature. Unbound primary antibodies were removed before incubation with species-matched secondary HRP-labeled polymer antibodies (Dako). Chromogen 3,3′-diaminobenzidine (Dako) was used as substrate for color development. Slides were counterstained with hematoxylin before dehydration and mounted with DPX (Sigma). For fluorescent immunodetection, species-specific secondary antibodies conjugated to Alexa Fluor 488/555 were used instead of HRP-labeled polymer antibodies. Sections were washed, counterstained with DAPI (100 ng/ml), and mounted using FluorSave (Calbiochem). For experiments in which goat primary antibodies were used, 5% BSA in PBS was substituted for 10% goat serum. Images were acquired on a Zeiss Axioimager microscope (for bright-field imaging) or an Olympus FluoView FV1000 (for fluorescent antibody detection).