Alexa fluor 594 conjugated anti mouse secondary antibody

FTC-133 cells were seeded on coverslips in a 12 well plate at an appropriate density of 60% confluency. 24 hours later the cells were fixed with 4% paraformaldehyde. Then cells were permeabilized with 0.3% tween 20 and blocked with 1% Bovine Serum Albumin (BSA). Primary antibody for ER α (1:100, Santa Cruz) was applied to cells overnight at 4°. Then the cells were incubated with Alexa Fluor 594 conjugated anti-mouse secondary antibody (Invitrogen) 1:400 for one hour followed by Hoechst 3334 incubation to stain the DNA. Coverslips were mounted on glycerol. Confocal microscope was used to analyse the images. Images of cells expressing ER α were acquired at 20X magnification.

BrdU (bromodeoxyuridine) assay was carried out to measure the amount of proliferating cells wherein cells were incubated with BrdU (Sigma, St. Louis, MO) solution for 1 h at 37°C. Cells were washed with PBS and fixed in 4% para-formaldehyde. Permeabilization in PBS containing 0.1% triton was followed by incubation with 1 (N) HCl on ice for 10 mins and 2 (N) HCl at RT for 10 mins. Phosphate citric acid buffer pH 7.4 was added and cells were incubated at RT for 10 mins prior to washes with PBS containing 0.1% triton. Cells were blocked in 5% bovine serum albumin for 1 h at 4°C and incubated overnight with anti-BrdU (1:500) (Cat #RPN202, RRID : AB_2314032, GE Healthcare, Buckinghamshire, UK) at 4°C. Following day, cells were incubated with Alexa Fluor 594-conjugated anti-mouse secondary antibody (Cat #A-11032, RRID : AB_2534091, 1:1000) (Invitrogen, Carlsbad, CA) for 1 h at RT. Cells were washed with PBS and nuclei were counterstained with DAPI. Cells were mounted on slides and observed under fluorescence microscope (Nikon-Eclipse-Ti-S, Tokyo, Japan). The number of BrdU positive cells was counted and graphically presented relative to the control.

Cells were plated in 96-well plates (4 × 10³/well) then cultured in normoxia or hypoxia. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% triton-X 100 in PBS for 10 min, blocked with PBS containing 5% FBS, and incubated with antibodies against AQP1, AQP5 (1:100, Millipore), or N-Cadherin (1:800 BD Transduction Laboratories) at 4°C overnight. For negative control, isotype IgG under similar conditions was used. After washing, the cells were incubated with Alexa fluor-488 conjugated anti-rabbit or Alexa-fluor-594 conjugated anti-mouse secondary antibody (1:50, Invitrogen) and following washing imaged using a fluorescence microscope (Nikon, Japan).