Ca 94010

For immunofluorescence analysis, 25 μm serial cryosections were obtained using a CM3050 cryostat (Leica Microsystems, Wetzlar, Germany). After incubation in a blocking buffer (1 × PBS/1% bovine serum albumin/0.3% Triton X-100) for 1 hr at room temperature, sections were incubated with the following primary antibodies overnight in PBS at 4°C: mBDNF (ab75040, Abcam), pTrkB (ab131483, Abcam), TH (sc25269, Santa Cruz Biotechnology, Inc), DAT (sc32259, Santa Cruz Biotechnology, Inc), BrdU (MCA2483, Bio-Rad, Hercules, CA, USA), NeuN (ABN78, Millipore Corporation), and Iba1 (019–19741, Wako Chemicals, Richmond, VA, USA). Sections were washed with PBST and incubated with fluorescent secondary antibodies (A11001, A11007, or A11037, Invitrogen, Carlsbad, CA, USA; CA94010, Vector Laboratories, Inc, Burlingame, CA, USA) for 2 hr at room temperature. Sections were mounted onto slides using a mounting medium (H-1200, Vector Laboratories, Inc) and imaged using a fluorescence microscope (Carl Zeiss Imager M1, Carl Zeiss AG, Oberkochen, Germany). Immunofluorescence was analyzed using the IMT i-Solution Inc 10.1 (17TH-5989 Walter Gage Rd., Vancouver, BC, CA) image analysis software.

For immunostaining, intestinal tissues were fixed in 4% paraformaldehyde in PBS at 4°C for overnight and then were embedded in optimal cutting temperature compound (OCT) (Sakura Finetek). 7-µm-thick frozen sections were boiled in 50 mM sodium citrate buffer solution (pH=6.0) for antigen retrieval, then were blocked using 1% BSA in PBS at room temperature for 1 hour. Subsequently, the sections were incubated with primary antibodies in PBS with 1% BSA at 4°C for overnight, and then incubated with secondary antibodies in PBS with 1% BSA at room temperature for 1 hour. The primary antibodies included rabbit anti-EpCAM (1:200; ab71916; Abcam), rabbit anti-mouse IgA secondary antibody (1:1000; NB7506; Novus) and rabbit anti-Claudin-7 (1:200; 34-9100; Thermo Fisher Scientific, Inc.). Immunohistochemical analysis was performed with biotin-conjugated goat-anti-rabbit secondary antibody (JAC-111-065-144, Jackson ImmunoResearch), HRP-ABC complex (CA 94010, VECTASTAIN) and DAB (LF778, DOJINDO Laboratories), and immunofluorescence analysis was performed with Alex Fluor 488-labeled secondary antibodies (Invitrogen). The immunostaining images were observed using the PerkinElmer Automated Quantitative Pathology System.