Alexa fluor 594 conjugated goat anti rabbit igg

Cells were pre-seeded on laminin-coated 8-well slides (Corning). After being treated with AREG, TGF-β1, inhibitors or siRNA-mediated knockdown, the cells were fixed and incubated with rabbit anti-VE-cadherin antibody (diluted at 1:500) overnight at 4°C. The cells were then thoroughly washed and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Abcam) for further 2 h at 4°C. After thoroughly washing again, the nuclei were stained with DAPI solution (1 μg/ml) for 5 min. Cell slides were maintained in the dark until images were taken under a fluorescence microscope. The fluorescence intensity in each field in the slides was quantified using ImageJ (Bethesda Maryland).

C2C12 transfected with 5 µg of pHA-Glut4-GFP plasmid and 10 µg of either pCMV-DNAJB3 or pCMV were plated on glass-bottom dishes. After stimulation with 100 nM of Insulin (Sigma Aldrich, St. Louis, MO), they were fixed with 4% paraformaldehyde without permeabilization and subjected to HA staining using a rabbit monoclonal anti-HA antibody (Rockland, Limerick, PA) followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK). The Alexa Fluor 595/GFP ratio was determined by quantitative fluorescence microscopy as described previously^{27 (link)}. To avoid cell selection bias fields, cells expressing the HA–Glut4-eGFP were randomly chosen in the GFP channel blinded to the expression of HA–Glut4-GFP on the plasma membrane (Alexa Fluor 594 channel). Images were collected in both GFP and Alexa Fluor 594 channels. To optimize the dynamic range of the assay, exposure times for the channels were independently set to maximize the signal while minimizing the number of cells with expression levels above saturation. Once set for each channel, all images in that channel were collected at the same exposure. The fluorescence intensities of GFP and Alexa 594 were quantified at the single-cell level. Mock- transfected cells were used in parallel to correct for fluorescence resulting from non-specific binding of the primary and/or secondary antibodies.

5 × 10⁵ DF-1 cells containing GJA8 or mutant GJA8Ala69Thr plasmid or vector transgene were cultured for 48 h on glass coverslips. Then the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA), permeabilized for 5 min in 0.5% Triton X-100, blocked for 1 h with 3% bovine serum albumin (Sigma-Aldrich) at room temperature. Subsequently, cells were incubated overnight at 4°C with anti-GJA8 (1:100, ab222885, Abcam), and further incubated for 1 h at room temperature with Alexa Fluor® 594-conjugated goat anti-rabbit IgG (1:500, ab150080, Abcam). A confocal microscope (Carl Zeiss AG, Oberkochen, Germany) was used to observe protein expression and subcellular localization.

Ki-67 expression was determined as a marker of cell proliferation. For this purpose, hDPCs were incubated on an 18 mm coverslip for 24 h in the presence of 100 ppm DA-5512 or 1 μM MXD. The cells were then washed twice with PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 37°C, permeabilized with 0.25% triton X-100 in PBS for 5 min, and blocked with 1% BSA in PBS for 30 min. The cells were incubated with an anti-Ki-67 antibody (1 : 100 dilution) (Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1 : 200 dilution) for 1 h at room temperature. The hDPCs were then washed and counterstained with 1 μg/mL DAPI for 5 min and mounted on a glass slide. Images of fluorescence were acquired using a confocal laser scanning microscope (TCS SPE; Leica Microsystems GmbH, Wetzlar, Germany).