Cy3 conjugated anti mouse igg

Paraffin blocks of the biopsied specimens from the hairless patches of 9 patients with AA (type 1, n=3; type 2, n=3; type 3, n=3; and type 4, n=0) were cut into 4-µm thick sections and incubated at 58℃ overnight. The paraffin sections were deparaffinized with 100% xylene two times for 10 minutes each and washed with graded concentrations (100%, 90%, 70%, and 50%) of ethyl alcohol, followed by application of 0.3% PBST solution for 10 minutes, a wash with PBS solution, and then application of 10% blocking solution for 60 minutes. The following primary antibodies were used: mouse monoclonal anti-CCR5 antibody (clone 45549; R&D Systems, Abingdon, UK) for the Th1 fraction, and rabbit polyclonal anti-CCR6 antibody (clone ab78429; Abcam, Cambridge, UK) for the Th17 fraction. These primary antibodies were applied to slide sections at 4℃ and incubated overnight. After slide sections were washed with PBS solution, Cy3-conjugated anti-mouse IgG (Invitrogen, CA, USA), Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen) or Oregon Green 488-conjugated anti-rabbit IgG (Invitrogen) were applied as secondary antibodies at 22℃ for 3 hours. Subsequently, after washing with PBS solution, the slides were observed using confocal microscope (LSM 510; Carl Zeiss, Jena, Germany) and photographed.