Figure 2
Lung Biopsy with pathology of AFOP. (a) Arrow shows fibrin ball (b) HO-1, TNF-α and controls at 25x and 100x. Samples were deparaffinized and rehydrated. Ag retrieval was performed with 0.01 M citrate buffer (pH6.0) for 20 min. Then endogenous peroxidase was blocked with 3% H2O2/PBS for 5 min. Tissue samples were blocked in 1% normal swine serum (NSS) for 15 min, before incubation with primary antibodies for TNFα (clone 2C8, Novus Biologicals, Littleton, CO, USA) 670 μg/mL or HO-1 (clone HO-1-1, Enzo Life Sciences, Farmingdale, NY, USA) 20 μg/mL overnight in 1% normal swine serum/PBS. Next day, sections were stained with biotinylated secondary goat anti mouse Ab (1:1500, Millipore, Darmstadt, Germany) for 1 h followed by streptavidin/HRP solution (Vector laboratories, Burlingame, CA, USA) 1:200 for 45 min. Samples were stained with DAB chromogen (Dako, Carpinteria, CA, USA) and counterstained with Mayer’s hematoxylin. Negative staining with isotype Ab run in parallel.
