Anti pro spc

Left lung tissues were fixed with 10% formalin overnight and paraffin embedded. Tissues were sectioned and stained with hematoxylin and eosin (H&E) according to standard protocols [32 (link)]. The severity of lung emphysema was assessed by measuring the mean linear intercept (Lm) [33 (link)]. The distance in airspace size between the alveolar walls was calculated by drawing equally distributed horizontal lines across each alveolus from wall to wall and recording the length for each measurement. Five random microscopic fields within each lung section were observed [34 (link)]. For IF analysis, the paraffin-embedded sections were deparaffinized and then blocked with 4% fetal bovine serum in PBS with 0.2% Triton X-100 for 2 h at room temperature. Sections were then incubated overnight at 4 °C with primary antibody as follows: anti-pro-SPC (Millipore, 1:4000), anti-AQP-5 (Millipore,1:800), and anti-GFP (Millipore, 1:750). Sections were incubated with secondary antibody (Alexa Fluor® 488 conjugated goat anti-mouse IgG or Alexa Fluor® 647 conjugated goat anti-rabbit IgG) for 1 h at RT. After washing with PBS, the slides were stained with DAPI for nuclear counterstaining and mounted with FluoreGuard Mounting Medium (Biosystems).

Sections (4 μm) were treated with 10% hydrogen peroxide, blocked with 2.5% normal horse serum, and incubated with anti-Kir7.1 [[5 (link)], 1:15,000] or anti-pro-SPC (1: 2,000; Millipore) overnight at 4°C. Detection of antigen/antibody complexes was performed with ImmPRESSTM (Peroxidase) Polymer Anti-Rabbit Ig Reagent (Vector Laboratories, USA). The reaction was visualized using Vector SG Peroxidase (HRP) Substrate Kit (Vector Laboratories, USA). In controls the incubation with primary antibody was omitted. Sections were counterstained with Vector Nuclear Fast Red (Vector Laboratories, USA).