Lung Tissue Immunofluorescence Analysis

Immunofluorescence staining was done as described before (2 (link)). FFPE lung sections were cut, immersed in xylene, and then hydrated in alcohol and PBS. Antigens were unmasked using DakoCytomation target retrieval solution (Dako), and non-specific binding was blocked by adding 5% (vol/vol) normal donkey serum and Fc block (BD). Avidin was used to neutralize endogenous biotin, followed by incubation with biotin (Sigma-Aldrich). Sections were then probed with anti-B220 (clone RA3-6B2, BD) to detect B cells. For analysis of B-cell follicles, follicles were outlined with an automated tool of the Zeiss Axioplan 2 microscope (Zeiss), and total area and average size were calculated in squared microns.