Alexa fluor 633 conjugated secondary antibodies

Cells were seeded in 12-well plates containing glass coverslips, then cultured and treated with EGF as described above for 72 h. After washing with PBS three times, cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized in freshly prepared 0.15% triton X-100 in PBS for 10 min, blocked in 3% BSA/PBS at room temperature for 1 h, and incubated with anti-E-cadherin (BD Biosciences), anti-Vimentin (abcam), anti-Slug (Cell Signaling Technology), anti-Anxa2 (Santa Cruz Biotechnology), anti-STAT3 (Cell Signaling Technology), and anti-p-STAT3 (Y705, Cell Signaling Technology) antibodies in a humid chamber at 4°C overnight. After repeated washing with PBS, cells were stained with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 633 conjugated secondary antibodies (Invitrogen) at room temperature for 1 h, followed by staining nucleus by DAPI. Finally, the coverslips were mounted with Mowiol-based anti-fading medium and visualized under a laser scanning confocal microscope (Leica TCS SP5).

Vibratome sections (60 μm thick) and retinal flat mounts were stained with rabbit anti-calretinin (1:1000, Millipore), goat anti-ChAT (1:500, Millipore), rabbit anti-recoverin (1:1000, Millipore), rabbit anti-PKARIIβ (1:500, BD Bioscience, San Jose, CA), rabbit anti-TH (1:1000, Millipore), rabbit anti-HCN4 (1:500, Neuromab, Davis, CA), rabbit anti-VIP (1:1000, Immunostar, Hudson, WI), mouse anti-melanopsin (1:1000, Advanced Targeting Systems, San Diego, CA), mouse anti-PKCα (1:500, Sigma, Saint Louis, MO), and mouse anti-Znp1/SytII (1:1000, ZIRC, Eugene, OR) for 1 (vibratome slices) or 5 days (flat mounts) at 4°C. The tissue was then washed in PBS (3 × 30 min), incubated with DyLight 405- (1:100, Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488-, Alexa Fluor 568-, and/or Alexa Fluor 633-conjugated secondary antibodies (1:1000, Invitrogen, Grand Island, NY) for 2 hr at RT (vibratome slices) or 2 days at 4°C (flat mounts), washed again in PBS (3 × 30 min), and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for confocal imaging.

HMVEC-L were incubated in ice-cold, low-serum (1% FBS) EGM-2MV medium for 30 min before inoculation with ANDV (MOI = 30) for 1 h at 4°C. The cells were washed with ice-cold PBS, and given ice-cold, low-serum EGM-2MV media containing 10 kDa dextran (250 μg/mL) or transferrin (100 μg/mL) conjugated to AlexaFluor 488 (Life Technologies); the cells were then incubated for 20 min on ice. Cells were transferred to 37°C for 15 or 30 min, and then washed 3 times with ice-cold 2 mM PBS glycine (pH 2) before fixing cells with 4% paraformaldehyde in PBS, permeabilizing in 0.1% Triton X-100 in PBS, and imaging for immunofluorescence. Briefly, cells were stained with polyclonal anti-ANDV primary antibodies and AlexaFluor 633-conjugated secondary antibodies (Life Technologies), and cell nuclei were counterstained with DAPI (1 μg/mL). The resulting coverslips were examined by confocal microscopy.