For immunofluorescent

staining, after deparaffinization a

For immunofluorescent

staining, after deparaffinization and rehydration, the antigens were retrieved by heating in citrate buffer (10 mM, pH 6.0) for anti-AQP4 antibody (121°C, 3 min). Nonspecific binding was Selleck Regorafenib blocked by a blocking reagent (Protein Block, Dako Japan, Tokyo, Japan). Sections were incubated overnight with the primary antibodies at 4°C. After rinsing in Tris-buffered saline with Tween 20 (TBS-T), the slides were incubated with Alexa Flour 488 donkey anti-rabbit IgG (1:1000, Life Technologies, Carlsbad, CA, USA). For double staining, after rinsing in TBS-T, anti-H+/K+-ATPase was digested with proteinase K solution (Dako Japan) for 4 min at room temperature. Subsequently, blocking reagent was applied for blockade of nonspecific binding, and then sections were incubated for overnight with anti-H+/K+-ATPase α subunit antibody. After rinsing in TBS-T, the slides were incubated with Alexa Fluor 568 goat anti-mouse IgG (1:1000, Molecular Probes). Total RNAs of tissue specimens from each stomach tissue specimens were extracted by using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). RNA was converted into cDNA using the PrimeScript RT reagent

kit (Takara, Ohtsu, Japan). The cDNA was used for quantitative PCR analysis with Dice (Takara) using SYBR Premix Ex TaqII (Takara). The mRNA expressions of mouse AQP4, H+/K+-ATPase α subunit, Shh, TFF2,

and Selleck Tamoxifen β-actin were measured. The primer sequences are shown in Table 1. Data for each gene were normalized to the expression level of β-actin. All data were expressed as mean ± standard error. Statistical significance of the differences between two groups was evaluated using unpaired Student’s t-test. All statistical analyses were performed using the SPSS Statistics version 20.0J software for Windows (SPSS Japan, Tokyo, Japan). A two-sided P value of < 0.05 was considered as denoting statistical significance. To investigate the progression of gastric lesion, HE staining was performed (Fig. 1a,b). The gastric mucosa in the H2R knockout mouse showed glandular hyperplasia with multiple cystic dilatations with increased mucous cells and parietal cells as well as SPEM. Also, Liothyronine Sodium the hyperplasia was more thickened with increase of cystic dilatation at the age of 40 and 60 weeks than at the age of 20 weeks (Fig. 1c,d). However, no gastric carcinoma lesion was observed even at the age of 95 weeks (data not shown). For the purpose of characterization of parietal cells along with vertical localization, fluorescent immunohistochemistry of H+/K+-ATPase and AQP4 was performed (Fig. 1e). In the wild type at the age of 20 weeks, H+/K+-ATPase-positive parietal cells were diffusely observed in the gastric mucosa, whereas AQP4-expressing parietal cells were mainly localized in basal area of the mucosa.

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