Lsm 510 confocal microscopy

HEK293 cells plated on coverslips and transfected with control or Sod1 siRNA (72 h), were fixed using 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min and blocked with 10% FBS for 1 hour. The coverslips were then incubated with the β-catenin antibody for 2 h, followed by Cy3-conjugated secondary antibody for 1 hour. 4′, 6-diamidino-2-phenylindole (DAPI,1:10,000 dilution, Sigma) was used for nuclear staining. The mounted coverslips were imaged using Zeiss LSM510 confocal microscopy to assess cytoplasmic versus nuclear localization of β-catenin. Results were expressed as percent localization relative to the total number of DAPI positive cells. At least 10 fields were imaged from each condition from 3 independent experiments.

Cell monolayers were washed thrice with 500 μL of HBSS after PIP peptides treatments, then washed once with the same volume of ice-cold MeOH prior to being fixed by placing in precooled MeOH at −20°C for 1 h. Cell monolayers were then re-hydrated by incubation in phosphate buffer saline (PBS) for 15 min at room temperature (RT) before being blocked with 10% (v/v) FBS in PBS for 1 h at RT. Monolayers were then washed thrice with PBS that followed by a 1 h incubation at RT with secondary antibodies-conjugated to Alexa Fluor^® 546 or Alexa Fluor^® 488 (Invitrogen, UK). Cell monolayer were then washed thrice with PBS and mounted on microscope slides, as described previously [20 ]. Rat intestinal tissues, exposed to PIP 640 via intraluminal injections (ILI) into rat mid-jejunum, were isolated as described previously [19 (link)]. Tissues were fixed with 4 % paraformaldehyde, embedded in paraffin, and allowed to set at 4°C overnight. Paraffin-embedded tissues were sectioned to obtain 5 μm slices that were placed onto glass slides. Tissues were then immune-stained with fluorescent antibodies, described above. Images were collected using a Zeiss LSM 510 confocal microscopy.