Immunohistochemical Detection of Chemokine Expression

HEK 293 cells transfected with CCL27, CCL28, or CCL25 were fixed with 20% ice-cold acetone, and air-dried for one day. Fixed cells were incubated with anti-V5 antibodies (Invitrogen) as primary antibodies at RT for 1 h, followed by monoclonal anti-rabbit/mouse immunoglobulins conjugated with horseradish peroxidase (HRP) (Dako, Carpinteria, CA, USA) as secondary antibodies at RT for another 1 h. The detailed procedure has been described previously [35 (link)]. After washing three times with Dulbecco’s Phosphate-Buffered Saline (DPBS) (Gibco), coloration was detected using the EnVision-DAB+ system (Dako, Santa Clara, CA, USA). Positive brown signals were identified under an inverted light microscope (Nikon, Tokyo, Japan).