Ebio500a2

Purified lymphocytes were counted using an automatic cell counter (Countess, BD Biosciences). Cells were stained with a 1/200 dilution of fluorochrome-conjugated antibodies against CD45 (clone 30F-11), CD3ɛ (eBio500A2), CD4 (RM4–5), CD8 (53-6.7), NK1.1 (PK136) and γδ TCR (eBioGL3) (eBioscience), and their populations were analysed by an LSR II flow cytometer (BD Biosciences) using software packages from CellQuest and FlowJo version 8.7.

Briefly, 4‐μm‐thick murine lung tissue slices were dewaxed and rehydrated in a graded series of ethanol. Next, their endogenous peroxidase activity was blocked and the sections were subjected to epitope retrieval, following which, they were incubated with primary antibodies against Anti‐mouse CD3e‐biotin (dilution 1:100 eBio500A2, eBioscience,Cal, USA), Anti‐rat GATA3 (dilution 1:50, ab110093, Abcam, Mass, USA), and Anti‐rabbit inducible T‐cell costimulatory (ICOS) (dilution 1:200, ab175401, Abcam, Mass, USA). Bound biotinylated antibodies were detected using streptavidin‐AF555 conjugated secondary antibodies (S32355, Invitrogen, Carlsbad, Cal, USA), then anti‐GATA3 antibodies were detected using rabbit anti‐rat IgG‐FITC‐AF488 (A‐11006, Invitrogen, Carlsbad, Cal, USA). Finally, ICOS antibodies were detected using goat anti‐rabbit IgG‐647 secondary antibodies (A‐27040, Invitrogen, Carlsbad, Cal, USA). Sections were counterstained with 4’,6‐Diamidino‐2’‐phenylindole (62248, Invitrogen, Carlsbad, Cal, USA). Images were acquired under an Laser Scanning Confocal Microscope (LSM) 510 meta confocal microscope (Zeiss, Oberkochen,UK) and the Zeiss LSM software was used for image analysis.