Bx40 fluorescent microscope

Bladder tissue was fixed in 4% paraformaldehyde overnight, then dehydrated within graded ethanol and xylene prior to being embedded with paraffin. Tissue was then sectioned at 5 μm. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide in tris-buffered saline (TBST) for 20 minutes and washed 3X in TBST for 3′. Antigen retrieval was performed using antigen unmasking solution (Vector laboratories, CA). To minimize non-specific anti- body binding, sections were incubated for 60 min in 5% fetal bovine serum in TBST with 0.3% Triton X-100 (Sigma Aldrich, St. Louis, MO). Sections were incubated overnight at 4 °C with rabbit anti-mouse IL-33 at a 1:400 in blocking solution (Santa Cruz Biotech, Inc., CA). Following incubation, slides were washed three times in TBST for 3 min. Sections were then incubated for 60 min. with DyeLight633 conjugated secondary antibody (goat anti-rabbit IgG 1:3000 in blocking solution (Thermo Scientific, IL) at room temperature, sections were washed three times for 3 min in TBST. Coverglass was added after one to two drops of FluoMount-G (Southern Biotech, AL) premixed with Hoechst 33342 (Invitrogen, CA) at 1:200 dilution for staining cellular DNA. Negative controls included incubation with TBST in place of the primary antibody and no immuno- reactivity was observed. Images were obtained on an Olympus BX40 fluorescent microscope.

For ICC, 2–3 × 10⁵ cells were seeded onto glass coverslips in 6-well plates. After treatments, cells were washed twice wish PBS and fixed with ice cold 70% Acetone/30% methanol for 10 min. Fixed cells were blocked with 10% normal goat serum (Vector Lab, Burlingame, CA, USA) in 0.05% TBS-T for 30 minutes followed by addition of primary antibodies (CK5, mouse NCL-L-CK5, Leica Biosystems; β-catenin, 9587, Cell Signaling Technologies, 1:200) for 2 h, secondary antibodies (A11029, A11037, Invitrogen, 1:200) for 1 h, and counterstained with DAPI. Cells were imaged using the Olympus BX40 fluorescent microscope or Olympus FV1000 laser scanning confocal microscope. Individual cells were scored for membrane β-catenin coverage of 0–25% (low), 25–75% (medium), or 75–100% (high) in a blinded manner. IHC was performed as previously described (71 (link)) using the antibodies described above and imaged using the Olympus FV1000 laser scanning confocal microscope.