Fluorescently labelled secondary antibodies

Immunohistology was performed according to standard procedures on 5 μm paraffin sections, using Peroxidase Substrate kits (Vector) or fluorescently labelled secondary antibodies (Jackson). Antibodies used: p14^ARF (Abcam, ab3642), CC3 (Cell Signaling, #9661), Ki67 (Labvision, RM-9106), K14 (Progen, GP-CK14) and K15 (Santa Cruz sc-56520). For SA-β-Gal stains, 10-12 μm cryosections of OCT-embedded mouse skins were fixed in 0.5% glutaraldehyde for 15 min and stained as previously described36 (link). Bright field images were collected using an Olympus CX41 and DS-Fi1 camera and processed using NIS Elements software (Nikon). Fluorescent images were collected using an Olympus FV1000 confocal microscope. SA-β-Gal staining in bronchioles area was quantified using ImageJ, and by visual scoring in the skin. Greater than 10 microscopic fields were scored in each sample in all stains. Proliferating hair-follicle stem cells were scored by counting the number of K15⁺Ki67⁺ cells per follicle, in >15 fields in each mouse (1–3 follicles per field); values indicate mean numbers per individual mice.

Ultra pure Lipopolysaccharide (LPS) from E. coli 0111:B4, Lipoteichoic acid (LTA), Pam₃CSK₄, FSL-1, HKLM, polyI:C (HMW), FlTC-labelled polyI:C (HMW), R848, CpG and 5’ triphosphate double stranded RNA (5’ ppp-dsRNA) were purchased from InvivoGen (San Diego, USA), M-CSF, ELISA DuoSets and IFNβ antibodies were obtained from R&D Systems, (Abington, UK). Fluorescently labelled secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (PA, USA). hBD3 (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK) was from Peptides International, and cys-ser hBD3 and cys-ser-TAMRA hBD3 were from Almac (Almac Group Ltd, Craigavon, UK).

Antibodies to Zfh2 (1:400, rat polyclonal, [56 (link)]), Ci (1:500, rat monoclonal 2A1, deposited into Developmental Biology Hybridoma Bank by R. Holmgren [57 (link)]), Nubbin (1:50, mouse monoclonal 2D4, deposited into Developmental Biology Hybridoma Bank by Michalis Averof), cleaved caspase Dcp1 (1:100, rabbit polyclonal, Cell Signaling #9578S) and fluorescently labelled secondary antibodies (1:200, Jackson) were used (see also S1 Table). In all experiments, wing discs were dissected in PBS, fixed in 4% para-formaldehyde in PBS for 30 min, and washed three times PBTx (0.1% Triton X-100). For antibody staining, the discs were washed in PBS instead of PBTx after the fixing step, permeabilized in PBTx with 0.5% Triton X-100 for 10 min and rinsed in PBTx. The discs were blocked in 5% Normal Goal Serum in PBTx for at least 30 min and incubated overnight at 4°C in primary antibody in block. The discs were rinsed thrice in PBTx and incubated in secondary antibody in block for 2 h at room temperature. Stained discs were washed in PBT. The discs were counter-stained with 10 μg/ml Hoechst33342 in PBTx for 2 min, washed 3 times, and mounted on glass slides in Fluoromount G (SouthernBiotech).