Ab63982

Immunofluorescence was carried out as previously described. Generally, the prepared sections underwent several steps including antigen retrieval, permeabilization, blocking, immunostaining, and counter staining sequentially. Finally, all the slides were mounted and analyzed via a fluorescence microscope (Olympus BX60). The primary antibodies of Ki67 (ab16667), γ-H2AX (ab2893), and defensin 1 (ab63982) were bought from Abcam company.

IHC labelling was achieved with the use of the standard Biotin—Streptavidin method to detect: HBD-2 (ab63982, working dilution 1:500, Abcam, Cambridge, UK), HBD-3 (orb183268, working dilution 1:100, Biorbyt Limited, Cambridge, UK), HBD-4 (ab70215, working dilution 1:100, Abcam, Cambridge, UK), IL-10 (orb100193, working dilution 1:600, Biorbyt Limited, Cambridge, UK), and LL-37 (orb88370, working dilution 1:100, Biorbyt LLC, St Louis, MO, USA) [53 (link),54 (link)].
The sample slides were analysed by light microscopy using non-parametric evaluation, which included grading of positively stained tracheal hyaline cartilage cells in the visual field. The results were labelled as follows: 0—no positive cells; 00/+—scant number of positive cells; 0/+—occasional positive cells; +—few positive cells; +/++—few to moderate number of positive cells; ++—moderate number of positive cells; ++/+++—moderate to numerous positive cells; +++—numerous positive cells; +++/++++—numerous to abundant positive cells; ++++—abundant number of positive cells [55 (link)].
The extracellular matrix was also analysed in Bismarck brown and PAS staining for neutral and acidic glycosaminoglycans, and the results were labelled accordingly: -—no positive ground substance; ±—partially stained ground substance; +—stained ground substance.

FFPE tissue sections (3.5 μm) were deparaffinized and rehydrated for immunohistochemical staining. Slides with tissues sections were incubated for heat‐induced antigen retrieval in Dako Target Retrieval Solution Citrate pH 6.0 (Dako S2369) or Dako Target Retrieval Solution pH 9,0 (Dako S2367) for 30 minutes in a steamer. The staining was then performed manually at 4°C antibody incubation using the Dako REAL™ Detection System, Peroxidase/AEC, using monoclonal antibodies directed against: HBD2 (1:400; #ab63982, Abcam Cambridge, U.K.), HBD3 (1:100; #LS‐B86, LSbio Seattle, WA), psoriasin (1:300; #MA1‐91555, Thermo Fisher Scientific, Pittsburgh, PA), RNase7 (1:50; #ab154143, Abcam Cambridge, U.K.) and LL37 (1:50; #63982, Abcam Cambridge, U.K.). Images of stainings were acquired with a DP71 digital camera (Olympus, Vienna, Austria), attached to an Olympus BX51 microscope.