Biotinylated anti cd45.1 clone a20

Adult thymus tissues from host mice were frozen, 7-μm sections were cut, and then fixed in acetone and stained with the following Abs: the mTEC marker ERTR5 (a gift from Dr. W. van Ewijk, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan), detected with Alexa Fluor 594 goat anti-rat IgM (Invitrogen), biotinylated anti-CD45.1 (clone A20; eBioscience), detected by Streptavidin Alexa 647 (Invitrogen), FITC anti-CD45.2 (clone 104; eBioscience), followed by rabbit anti-FITC (Invitrogen), and donkey anti-rabbit 488 (Invitrogen), biotinylated anti-CD8 (clone YTS156.7.7; BioLegend), detected by Streptavidin Alexa 488 (Invitrogen), and purified anti-CD4 (clone GK 1.5; eBioscience), conjugated to Alexa 647 (Invitrogen). Images were obtained using a LSM 780 microscope and analyzed using LSM software. (Zeiss). For confocal quantitation, a minimum of three separate thymus sections at least 10 sections apart were analyzed for each mouse. Calculation of cell frequency within a defined area was performed as previously described (34 (link)), where square fields of 100 × 100μm were arbitrarily set within either medullary or cortical regions, at least 100 μm from the CMJ.