The procedure has been described in our previous publication [46 (link)]. In brief, slides were deparaffinized in xylene and gradually re-hydrated through 100% alcohol to distilled water. The rehydrated slides were subjected to heat-induced antigen retrieval in either 10 mM sodium citrate buffer (pH 6.0) or 10 mM Tris buffer (pH 9). After treating with 3% hydrogen peroxide followed by PBS wash, slides were incubated with protein block serum free solution (Agilent DAKO; Santa Clara, CA, USA) for 10 min at room temperature. After the primary antibody was incubated on the slides, the ImmPRESS Polymer Detection Kit (Vector Laboratories; Burlingame, CA, USA) was used according to the manufacturer’s instructions. For immunofluorescent staining, instead of using the ImmPRESS Polymer Detection Kit, Alexa-Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) corresponding to the primary antibody species were applied to the slides according to the manufacturer’s instructions. Images were collected using the Aperio VERSA tissue scanner with ImageScope software v12.4.3.5008 (Aperio; Sausalito, CA, USA).
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