B220 alexa fluor 647 ra3 6b2

Fresh spleens or LNs were snap-frozen in Tissue-Tek optimum cutting temperature (O.C.T.) compound (Sakura Finetek). Cryosections of 10 μm were cut, fixed in 4% of paraformaldehyde for 10 min followed by another 10 min incubation in pre-chilled acetone at −20 °C, and then washed three times with PBS for immunostaining. Following incubation in blocking buffer (CAS-Block, Invitrogen), the samples were incubated with fluorescence-labeled antibodies overnight at 4 °C. The primary antibodies used are anti-mouse CD8-Alexa Fluor 594 (53-6.7), anti-mouse B220-BV510 or B220-Alexa Fluor 647 (RA3-6B2 for both fluorochromes), and anti-mouse CD45.2-Alexa Fluor 488 (clone 104, all from BioLegend). Confocal images of cryosections were acquired using a Zeiss LSM710 confocal fluorescence microscopy and were processed with Imaris software (Bitplane).