Fluoview 500 9 71 confocal microscope

For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 30 minutes, washed 3 times with PBS, and permeabilized by incubation in PBS with 0.1% Triton X-100 for 10 minutes. After blocking in PBS with 10% goat serum and 0.1% Triton X-100 for 1 hour at room temperature, cells were incubated with the primary antibodies (anti-FXR, Abmart, China) monoclonal mouse antibody and anti-β-Catenin rabbit antibody (Cell Signaling) at 4°C overnight, followed by incubation with the secondary antibodies (Alexa488-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) and Alexa 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) at room temperature for 2 hours. Coverslips were mounted onto glass slides with anti-fade mounting medium with DAPI (Vector Laboratories) and visualized with an Olympus FluoView 500 IX71 confocal microscope.

Immunofluorescence assay was carried out according to the standard protocols. The specific biomarkers of CAFs, α-SMA, FSP, vimentin and CD44 induced by the CAFs in SW48 and LOVO cells were determined by immunofluorescence assay. Cells planted on the glass slide and frozen sections were washed with PBS, following by fixing with 4% paraformaldehyde for 30 min and then permeabilized in 0.1% Triton X-100 for 25 min. After being blocked with goat sera at room temperature for 1 h, cells and sections were incubated with antibody against α-SMA (1:100; proteintech, China), vimentin (1:100; proteintech, China), FSP (1:100; proteintech, China) or CD44 (1:100; proteintech, China) overnight at 4 °C, rinsed with PBS, incubated with suitable Dylight 594 red-conjugated or Alexa Flour 488 green-conjugated secondary antibodies (1:500; Multi-Sciences Biotech Co. Ltd, Hangzhou, China) for 1 h at room temperature, washed with PBS thrice and costained with 10 μg/ml 40,60-diamidino-2-phenylindole (DAPI) (Sigma, USA) for 10 min and finally observed and imaged with an Olympus Fluoview 500 IX 71 confocal microscope (Tokyo, Japan). Images were digitally recorded at the same magnification and time of exposure.

The cochlea and Auditory Cortex sections were thawed and dried for 30 min at room temperature, permeabilized with 0.1% Triton X-100(Sigma) in 50mM PBS for 20 min, washed 3 times and blocked with 10% goat serum for 1 h at room temperature. After overnight incubation with the primary antibody, rabbit anti-CaV1.3 calcium channel polyclonal antibody (1:50; Alomone labs, Israel) or mouse anti-Myo7a antibody (1:100; Santa Cruz, CA) at 4°C the sections were washed three times with PBS and incubated using Dylight 488 conjugated goat anti-rabbit IgG or Dylight 594 conjugated goat anti-mouse (1:500; Multi-Sciences, Hangzhou, China) for 1 h at room temperature. The sections were then washed with PBS and costained with 10μg/ml DAPI (Sigma, USA) for 20 min. After finally washing with PBS, the slides were covered with glass cover slips and observed under an Olympus Fluoview 500 IX 71 confocal microscope (Olympus, Tokyo, Japan). The images were digitally recorded at the same magnification and time of exposure. For hair cells examination, the surface of the OC was prepared by removing the otic capsule and tectorial membrane of the fixed cochlea. The dissected specimens were labeled with anti-CaV1.3 and DAPI before observation.