R d cell and tissue staining kit hrp dab system

Paraffin-embedded tissue sections were incubated in a 60°C oven for 1 hr and de-paraffinized in three changes of xylene, followed by 100% alcohol twice for 3 min each. Then the slides were transferred once in 95% and 70% alcohol, each for 3 min. A heat antigen retrieval step was used to unmask the antigenic epitopes. The remaining procedure was performed according to the manufacturer’s protocol (R&D Cell and Tissue Staining Kit HRP-DAB system, R&D Systems). Primary antibodies are listed in Supplemental Information. Biotinylated anti-mouse and anti-rabbit secondary antibodies are supplied by HRP-DAB system for Mouse (CTS002, R&D Systems) and Rabbit Kit (CTS005, R&D Systems). Double-sorted cells were plated onto a glass slide coated with poly-L-lysine (Sigma), and cells were allowed to attach overnight. The following day, cells were fixed in 4% paraformaldehyde, washed three times in 1× PBS and stained with primary antibody. Alexa-488-conjugated goat anti-mouse secondary antibody (A-11001, Invitrogen) was used followed by DAPI nuclear counterstain (H-1200, Vector Laboratories).