Anti pth2r

Protein isolation and transfer were performed as previously described (MacLeod et al., 2013 (link); Yamasaki et al., 2006 (link)). Anti-TIP39 (1 μg/ml; Santa Cruz, CA or Fitzgerald, MA), anti-PTHrP (1 mg/ml; Santa Cruz), anti-PTH2R (0.4 μg/ml; gift from Dr Usdin, NIMH), anti-glyceraldehyde-3-phosphate dehydrogenase (1 μg/ml; Fitzgerald), and antiβactin (1 μg/ml; Sigma-Aldrich, MO) antibodies were used for protein detection. IR-dye conjugated secondary antibodies (680 and 800 nm; 1:10,000) and an Odyssey imager and quantification software (LI-COR Biosciences, Lincoln, NE) were used for two-color western blotting to analyze both proteins on the same blot.

Immunostaining was performed by the manufacturer’s instructions (CST, Immunofluorescence General Protocol of IF-P). The skin tissue samples were fixed in 10% formalin for 24 hours. Five-micron paraffin sections were deparaffinized and rehydrated before heat-induced antigen retrieval was performed in 10 mM citrate buffer (pH 6.0). Anti-TIP39, anti-PTH2R, anti-Keratin14 (Santa Cruz), anti-FLG, anti-p63 (Thermo Scientific, IL), anti-PCNA (CST), secondary Alexa Fluor 488, or 594-conjugated antibodies (Life technologies) were used at 1 μg/ml. Epidermal thickness, FLG positive area, and PCNA positive cells were calculated as in previous reports (Gonzalez et al., 2003 (link); Marsella et al., 2013 (link)).