Ab51983

Parotids were collected at 1, 5 or 20 weeks after radiation therapy and fixed in 4% paraformaldehyde for histological or immunohistochemical detection. Samples were dehydrated with gradient ethanol and embedded in paraffin or frozen to obtain sections. Paraffin sections and frozen sections were subjected to immunohistochemistry (IHC) and immunofluorescent (IF) staining, respectively. These sections were incubated overnight at 4 °C with primary antibodies against AQP5 (1:200, ab78486), Ptch1 (1:200, ab51983), IL-6 (1:200, ab6672) (these 3 antibodies are from Abcam, Boston, MA, USA), ARG1 (1:100, A4923, Abclonal, Woburn, MA, USA), or VEGF (1:100, bs-1313R, Bioss, Woburn, USA). After being washed with PBS, appropriate secondary antibodies were incubated for 30 min at room temperature, and nuclei were counter-stained with DAPI for IF staining. The stained sections were imaged and quantified by investigators blinded to the treatments carried out in the experiment. At least two sections from 3 independent parotids were randomly chosen, and 2 random 200× fields of each section were imaged for quantification (n = 2 × 3 × 2 = 12).

Total RNA extraction, reverse transcription, and quantitative PCR (qPCR) were performed as reported [44 (link),45 (link)]. Primers for swine GAPDH, C1qA, TNF-α, IFN-γ, IL-4, IL-6, P53, AIF1, ADGRE 1, ITGAX, HGF, and ARG 1 were designed using Primer3 software (http://bioinfo.ut.ee/primer3/) and listed in Supplementary Table S1. For protein extraction, fresh parotid samples were homogenized in T-PER reagent containing protease inhibitors (Pierce, WA, USA). The concentration of protein was quantified using BCA, and the loading quantity was 25 μg per lane. Western blotting was done using primary antibodies against AQP5 (1:5000, ab78486), Ptch1 (1:5000, ab51983), TNF-α (1:5000, ab1793), IL-6 (1:200, ab6672), P53 (1:500, ab154036) (these 5 antibodies are from Abcam, Boston, MA, USA), Gli1 (1:1000, bs-1206R), F4/80 (1:1000, bs-7058R), AIF1 (1:1000, bs-1363R), and VEGF (1:100, bs-1313R) (these 4 antibodies are from Bioss, Woburn, MA, USA). All PCR and Western blot analyses were repeated at least three times.