Immunofluorescence Staining of Tissue Sections

Single and double immunofluorescence staining was performed as described by us already elsewhere^{32 (link),42 (link)}. In brief, deparaffinized tissue sections were heated in citrate buffer, pH 6 (Dako), for 20 min in a pressure cooker. Sections were then blocked (to avoid non-specific bindings) with ready to use 10% normal goat serum (Life Technologies, MD, USA) for 1 h at room temperature before incubating with primary antibody at 4 °C overnight. After washing in TBS-T for 10 min, the sections were incubated with Alexa conjugated secondary antibody (1 : 500 in TBS-T) at room temperature for 2 h. Sections were washed in TBS-T (2 × 10 min) and stained for 10 min with 0.1% Sudan Black in 80% ethanol to suppress endogenous lipofuscin auto-fluorescence. Finally, the sections were washed for 5 min in TBST and mounted with Vectashield mounting medium (Vector Laboratories) containing DAPI.