Prolong gold antifade mounting medium with 4 6 diamidino 2 phenylindole dapi

Tissues were fixed overnight in 10% formalin, de-paraffinised in xylene and then rehydrated in 70% ethanol. Antigen retrieval was performed in citrate buffer, pH 6.0, or Tris-EDTA, pH 9.0, for 30 min, and then blocked for 1 hour in 10% donkey serum diluted in Tris-buffered saline 0.1% Triton with 0.025% Tween-20 (TBST-TW). For immunofluorescence (IF), the primary antibodies were incubated in 1% bovine serum albumin (BSA) TBS-TW overnight at 4°C. Secondary antibodies (1:500) were incubated for 1 hour in 1% BSA with 0.025% Tween-20 (TBS-TW) at room temperature (RT) in the dark. For IHC, primary antibodies were incubated in 1% BSA Tris-buffered saline 0.1% Tween (TBS-T) overnight at 4°C. Slides were incubated in horseradish peroxidase (Cell Signaling, Danvers, Massachusetts) secondary antibody (1:300) for 60 min in 1% BSA TBS-T for 1 hour at RT. Slides were then incubated with diaminobenzidine (Vector, Burlingame, California) followed by counterstaining with haematoxylin (Leica, Buffalo Grove, Illinois). Dilutions of primary antibodies are listed in online supplemental table 2. Slides were mounted with ProLong Gold Antifade Mounting Medium with 4’, 6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were photographed on an Olympus BX53F microscope with an Olympus digital camera (Center Valley, Pennsylvania).