Immunohistochemistry and immunofluorescence staining of mouse tissue sections.

Slides were heated at 60°C for 30 minutes, deparaffinized with xylene, and then rehydrated through a descending ethanol series (100%–0%). A hydrogen peroxide/methanol solution was used to block endogenous peroxidase activity, and TBS plus 0.2% Tween 20 (TBST) plus 20% Aquablock was used to block nonspecific binding of antibodies. Slides were incubated overnight with primary antibodies diluted in TBST plus 5% BSA. Slides were washed with TBST and then incubated for 1 hour with secondary antibodies diluted in TBST plus 5% BSA. Antibody binding was detected with an ImmPACT DAB Peroxidase Substrate Kit (Vector, SK-4105). Slides were then dipped in hematoxylin and dehydrated, and coverslips were mounted with CytoSeal (Thermo Fisher Scientific, 8312-4).