Lung lobes were perfused with 10% neutral-buffered formalin and embedded in paraffin. For immunofluorescent staining, formalin-fixed lung sections were cut, immersed in xylene to remove paraffin, and then sequentially hydrated in absolute ethanol, 95% ethanol, 70% ethanol and water. Antigens were unmasked with a DakoCytomation Target Retrieval Solution (Dako, Carpinteria, CA) and non-specific binding was blocked with 5% (v/v) normal donkey serum and Fc block (BD Pharmingen). Endogenous biotin (Sigma-Aldrich) was neutralized by adding avidin followed by incubation with biotin. Sections were probed with anti-CD45R/B220 to detect B cells (Clone RA3-6B2, BD Pharmingen), anti-CXCL13 (AF470, R&D systems) and anti-CD3 to detect T cells (Clone M-20, Santa Cruz Biotechnology, Santa Cruz, CA). B-cell follicles were outlined with the automated tool of the Zeiss Axioplan 2 microscope (Carl Zeiss, Thornwood, NY) and average size in squared microns was calculated as described before12 (link).
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Lung Immunofluorescent Staining Protocol
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Corresponding Organization : Washington University in St. Louis
Other organizations : University of Rochester Medical Center
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Variable analysis
independent variables
- Perfusion of lung lobes with 10% neutral-buffered formalin
- Embedding lung lobes in paraffin
- Size of B-cell follicles (in squared microns)
- Formalin-fixed lung sections
- Immersion in xylene to remove paraffin
- Sequential hydration in absolute ethanol, 95% ethanol, 70% ethanol and water
- Antigen unmasking with DakoCytomation Target Retrieval Solution
- Blocking of non-specific binding with 5% (v/v) normal donkey serum and Fc block
- Neutralization of endogenous biotin by adding avidin and biotin
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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