Eclipse ni

Transbronchial biopsy slides underwent deparaffinization, rehydration, and antigen retrieval as described in Immunohistochemistry and imaging analysis. Slides were pretreated with protease. We had two probes designed to bind to the hypervariable CDR3-β segment of our most abundant clone in participants P1 and P8 at the time of ACR; probes were hybridized to our target mRNA and amplified per the manufacturer’s instructions. Slides were counterstained with 50% hematoxylin solution, washed with tap water, immersed in 0.02% ammonia water, and again washed with tap water before mounting. Images were captured at both 40× and 60× magnification using a Nikon Eclipse Ni and a digital camera (Hamamatsu Digital Camera C11440). Whole slides were scanned at 40× using a Zeiss Axio Scan.Z1, and positively stained cells were manually counted and determined to be close to the airways if they were within the intra-epithelial, subepithelial, or peribronchiolar regions of the lung.

5-μm sections of paraffin-embedded transbronchial biopsies were obtained from paraffin-embedded blocks maintained by the Pathology Department at the University of Pittsburgh. Slide deparaffinization was performed with >98.5% xylene followed by serial dilutions of ethanol. Antigen retrieval was performed at 95°C for 20 min in the presence of DAKO Target Retrieval Solution, pH 9 (Agilent), using a Decloaking Chamber NxGen (Biocare Medical). Primary antibody was stained overnight on an orbital shaker at 4°C. Secondary antibody was then applied for 1 h on an orbital shaker at room temperature (Table S3 includes list of all antibodies used). Slides were washed and stained with 1× DAPI for 5 min. Coverslips were then mounted with ProLong Gold antifade reagent (Invitrogen) and sealed with clear nail polish. Images were captured within 72 h with an epifluorescence microscope (Nikon Eclipse Ni) and a digital camera (Hamamatsu Digital Camera C11440). ImageJ was used to qualitatively analyze images and generate TIFF files from ND2.