Hrp dab cell tissue staining kit

Formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded series of ethanol, followed by antigen retrieval with citrate buffer (pH 6.0) for 20 min at 95°C. Sections were incubated with a primary antibody or an isotype control in a humidified chamber overnight at 4°C. Please refer to Supplementary Table S4 for a complete list of antibodies. 1) For immunohistochemistry: Sections were incubated with appropriate secondary antibodies using a HRP-DAB Cell & Tissue Staining Kit (R&D Systems) according to manufacturer’s instructions; 2) For immunofluorescence: Sections were incubated in Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature in the dark. Slides were then mounted using mounting medium containing 4,6-diamidino-2-phenylindole-2-HCl (DAPI) (Vector Laboratories Inc., Burlingame, CA). To detect in vivo cell apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using the In Situ Cell Death Detection Kit, TMR red (Roche Applied Science, Branford, CT) according to the manufacturer’s recommendations.