Rabbit anti mouse hrp conjugated secondary antibody

Single-cell suspensions from unchallenged or bleomycin-challenged mouse lungs were generated as described in the Flow cytometry section. After lysis of red blood cells, cell suspensions were depleted for leukocytes, endothelial cells, and epithelial cells using a mixture of anti-CD45–, anti-CD31–, and anti-EpCAM–coated magnetic beads, MS columns, and a MACS separator according to the manufacturer’s protocol (Miltenyi Biotec). After depletion, cell suspensions (enriched for CD45⁻,CD31⁻,EpCAM⁻ cells including fibroblasts) were washed three times in ice-cold PBS, and cells were lysed in lysis buffer (1% Triton X-100, 50 mM Tris, and 100 mM NaCl, pH 7.4) containing protease inhibitor cocktail III (1:200; Sigma-Aldrich). Protein concentrations in lysates were determined using a BCA kit (Bio-Rad). uPARAP was detected in a total of 3 µg protein from each lysate separated by SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. A 2% BSA blocking solution was applied before detection using 0.5 µg/ml anti-uPARAP mAb 2h9 as primary antibody and rabbit anti-mouse HRP-conjugated secondary antibody (1:3,000; Dako), both diluted in a 0.1% Tween-20 PBS solution. For loading control, 6 µg protein of each lysate was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining.