Fv1000 tirf microscope

Healthy GPR120^ΔIEC and WT 8 weeks old mice were fed for two weeks with cornmeal mush, then sacrificed by CO₂ and colon tissues embedded in OCT without any washing steps. Cryosections of 8 μm were fixed with Metacarnoy solution (75% Methanol + 25% Acetic Acid) for 4 h at room temperature, then washed in Ethanol (EtOH) and dried in the oven for 30 min. The hybridization area was marked with diamond cutter in order to localize it during the rest of procedure. The fluorescence in situ hybridization (FISH) assay was performed using an Alexa Fluor 555 (Invitrogen)–conjugated eubacterial probe (EU 622773). Each slide was stained with the bacterial probe diluted in freshly prepared Hybridization Buffer [Sodium Cloride (NaCl) 2 M; TRIS HCl 1 M; Sodium Dodecyl Sulfate (SDS) 10%; H₂0). The hybridization chamber was prepared and then loaded into DAKO machine overnight. Rubber cement was removed to wash slides in water-bath at 55 °C for 20 min. Washes in hybridization buffer + 2%BSA were performed to remove nonspecific hybrids. Colon sections were incubated with primary [Muc2 (Santa Cruz, B306.1; cat n° sc-59859), mouse anti-human/mouse, 1:25 dilution] and secondary (goat anti-mouse, 1:1000 dilution, Life Technologies) antibodies at room temperature for 1 h and then stained with DAPI. Images were acquired with an Olympus FV1000/TIRF microscope.