Histofine simple stain mouse max peroxidase

Routine morphological analysis was performed using lung tissue sections stained with hematoxylin and eosin (H&E) and the Masson-Trichrome (MT) method. Immunohistochemical (IHC) staining was performed on paraffin-embedded lung tissue sections. Briefly, paraffin sections were deparaffinized. Then, sections were subjected to heat-induced antigen retrieval (HIAR) in a 10 mM citric acid buffer (pH 6). Then, endogenous peroxidase activity was quenched with 3% hydrogen peroxide in PBS for 20 min and subsequently blocked with 10% goat serum for an additional 20 min. Sections were then incubated with either rabbit anti-club cell secretary protein (CCSP) antibody (EMD Millipore, Billerica, MA), rabbit anti-type I collagen antibody (Abcam, Cambridge, MA), or rabbit anti-claudin-10 antibody (Life Technologies, Carlsbad, CA). Next, the sections were incubated for 30 min with goat anti-rabbit IgG, which was conjugated with horseradish peroxidase (HRP) (Histofine Simple Stain Mouse MAX Peroxidase; Nichirei, Tokyo, Japan). Finally, detection of the target protein was performed using 3, 3’-diaminobenzidine (DAB) reagent.

Single-color immunohistochemical (IHC) staining was performed on paraffin-embedded lung tissue sections as follows. First, paraffin sections were deparaffinized. Then, sections were subjected to heat-induced antigen retrieval (HIAR) in a citric acid buffer (10 mM citric acid, pH 6) or Tris-EDTA buffer (10 mM Tris, 1mM EDTA, 0.05% Tween 20, pH 9.0). Then, endogenous peroxidase activity was quenched with 3% hydrogen peroxide in PBS for 20 min. Subsequently sections were blocked with PBS containing 5% bovine serum albumin (BSA) plus 0.25% Tween 20, and sequentially with 10% goat serum (diluted in PBS). Sections were then incubated with rabbit anti-Cldn10 antibody (Life Technologies, Carlsbad, CA) at 4°C overnight. After washing, the sections were incubated for 30 min with goat anti-rabbit IgG antibody, which is conjugated with horseradish peroxidase (HRP) (Histofine Simple Stain Mouse MAX Peroxidase; Nichirei, Tokyo, Japan). Finally, detection of the target protein was performed using 3, 3′-diaminobenzidine (DAB) reagent.

Male C57BL/6J adult mice (8 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). Mice were maintained under a 12-h light/dark cycle and fed ad libitum. All experiments were approved by the Animal Committee of Kyushu Dental University (No. 16–011). Collagenase L was purchased from Nitta Gelatin (Osaka, Japan); Fura 2-AM was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan); 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) was purchased from Life Technologies Corporation (Eugene, OR, USA); T16Ainh-A01 was obtained from Tocris Bioscience (Bristol, UK); the NKCC1 antibody (sc-21545) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA); the TMEM16A antibody (ab53213) was purchased from Abcam (Cambridge, UK); the AQP5 antibody (sc-9890) was purchased from EMD Millipore (Billerica, MA, USA); and the secondary antibody (Histofine Simple Stain Mouse MAX peroxidase) was purchased from Nichirei (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich Japan (Tokyo, Japan).