Ab76238

We deparaffinized the sections to water and placed in xylene for 20 min, which were performed three times. Slices were put in 100, 95, 85, and 75% ethanol in sequence for 5 min at each level. Slices were washed with distilled water for 5 min. The slices were immersed in citrate buffer (pH 6.0) and boiled in an electric furnace or microwave oven. After being cooled, slices were washed with 0.01 M of PBS (pH 7.2 ~ 7.6) for 3 min, which were performed three times. Slices were place in sodium borohydride solution at room temperature for 30 min. The sections were placed in Sudan black dye solution at room temperature for 5 min. Slices were blocked with 10% normal serum/5% bovine serum albumin (BSA) for 60 min. Slices were placed in appropriate first antibody, cAMP (1:50, rabbit, ab76238, Abcam, UK), MUC2 (1:50, rabbit, ab76774, Abcam, UK), Reg3γ (1:50, rabbit, ab233480, Abcam, UK), β-defensin (HBD-2) (1:50, rabbit, bs-1296r, Bioss, China), Claudin-1 (1:50, rabbit, 13050-1-AP, PTG), and ZO-1 (1:50, rabbit, 21773-1-AP, PTG), overnight at 4°C. Slices were incubated with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00013-2, Proteintech, USA) and incubated at 37°C for 90 min. Slices were stained in the nucleus with DAPI (Wellbio, China) working solution at 37°C for 10 min. Slices were stored in the dark or observed under a fluorescence microscope.

Western blotting analysis was carried out as reported previously by our lab (20 (link)). Briefly, after the total protein of soft palate tissues was extracted and separated, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane. Following washing, the membrane was incubated overnight at 4°C with primary antibodies against cAMP (1:1000, Abcam, ab76238), protein kinase A (PKA) (1:1000, Abcam, ab75991), cAMP-regulated guanine nucleotide exchange factor II (Epac2) (1:1000, Abcam, ab193665), rat sarcoma protein (Ras) (1:1000, Abcam, ab52939), c-Jun N-terminal kinase 1/2 (JNK1/2) (1:1000, Abcam, ab4821), Annexiv V (1:500, Abcam, ab14196), GAPDH (1:1000, Abcam, ab8245), and Tubulin (1:1000, Abcam, ab44928). After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at room temperature, followed by color development using a mixed solution. The image analyzer quantitative system was used to quantitatively determine the intensity of the target and reference proteins.