Bovine anti goat

Harvested mouse tissues were fixed in 2% paraformaldehyde for 2 h before being treated with 30% sucrose overnight for cryoprotection. The sucrose-treated tissue was embedded in OCT tissue-freezing medium and frozen in a 2-methyl butane liquid bath. Frozen blocks were processed, stained, imaged, and enumerated by QIM as described (Steinert et al., 2015 (link)). Tissues were stained with antibodies to the following markers: CD4 (GK1.5; BD Biosciences), CD90.1 (OX-7; BD Biosciences), CD45.1 (A20; BioLegend), B220 (RA3-6B2; BioLegend), CD8β (YTS156.7.7; BioLegend), and collagen-IV (goat anti-mouse polyclonal; Millipore). We counterstained with DAPI or SYTOX Green (Thermo Fisher Scientific) to detect nuclei. The following secondary antibodies were from Jackson ImmunoResearch: bovine anti-goat (polyclonal) and donkey anti-rat (polyclonal).

Western Blot analysis was performed as described previously with a modified lysis buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM β-glycerophosphate, 50 mM NaF, 0.5 μM NaVO3, and 1% [vol/vol] Triton X-100 supplemented with complete protease inhibitors (all were purchased from Sigma-Aldrich). Further, blots were blocked in 5% milk (in TBS–Tween 20), and primary antibodies were diluted in milk blocking solution. The following HRP conjugated secondary antibodies were all sourced from Jackson Immuno-Research, Monoclonal Mouse Anti-Rabbit (cat # 211-032-171;RRID:AB_2339149), Goat-Anti-Mouse (cat # 115-035-174; RRID:AB_2338512) and Bovine Anti-Goat (cat # 805-035-180; RRID:AB_23408074).