E cadherin primary antibody

VK-2 cells in keratinocyte serum-free medium (SFM) were grown on glass coverslips at 37°C and 5% CO₂ and infected with C. albicanstca17Δ/Δ or control and complemented strains at a multiplicity of infection (MOI) of 0.01 for 6 and 24 h, as previously described (8 (link), 72 (link)). A 1:100 dilution of an E-cadherin primary antibody (R&D, Minneapolis, MN) in 0.2% gelatin-PBS was then used to stain the VK-2 cells for 1 h. VK-2 cells were subsequently washed and incubated with an anti-rat Alexa Fluor 488 secondary antibody (Invitrogen, Waltham, MA) for 1 h. Coverslips were mounted on slides with an antifade mounting solution containing DAPI for nuclear visualization. Images were taken using a Zeiss Axio Imager M1 microscope with standard enhanced green fluorescent protein (eGFP) and DAPI filters and assessed for the presence of E-cadherin.

VK-2 cells growing on coverslips were infected with C. albicansent2Δ/Δ or control strains for 6 and 24 h in keratinocyte SFM medium at 37°C and 5% CO₂ as described previously (71 (link)). Cocultured C. albicans and VK-2 cells were fixed with 4% paraformaldehyde solution and incubated for 20 min. Next, the coverslips were washed with 1× PBS. The fixed cells were incubated with ammonium chloride for 10 min, followed by incubation with Triton X-100 for 5 min. Cells were then washed three times with PBS containing 0.2% gelatin at 5 min each. VK-2 cells were stained for 1 h with a 1:100 dilution of a E-cadherin primary antibody (R&D, Minneapolis, MN) in PBS containing 0.2% gelatin, washed, and incubated with an anti-mouse-green fluorescent protein (GFP) secondary antibody (Invitrogen, Waltham, MA) for another hour. Cover slides were mounted on slides with an antifade mounting solution containing DAPI. A Nikon Eclipse 80i microscope was used to visualize the cells using standard GFP and DAPI filters.

Western blotting was performed with a standard protocol as described in Kurien and Scofield (73 (link)) using protein lysates from VK-2 cells infected with C. albicanstca17Δ/Δ mutant or control and complemented strains for 6 and 24 h in keratinocyte SFM at 37°C and 5% CO₂. The blot was then hybridized overnight in 1× Tris-buffered saline casein blocking buffer (Bio-Rad, Hercules, CA) with a 1:500 dilution of an E-cadherin primary antibody (R&D, Minneapolis, MN) or 1:1,000 dilution of a tubulin antibody (Invitrogen, Waltham, MA) as a loading control. After washing, the blot was incubated with an anti-mouse horseradish peroxidase (HRP) secondary antibody (Invitrogen, Waltham, MA) for 1 h. Protein detection was performed using Clarity Max Western ECL substrates (Bio-Rad, Hercules, CA) and imaged with a ChemiDoc imaging system (Bio-Rad, Hercules, CA).