Cy3 conjugated goat anti rabbit igg antibody

GF-1 cells were first transfected with pcDNA3-CKB for two days and then infected with NNV for one day. After transfection and infection, GF-1 cells were rinsed three times with PBS and then were fixed in paraformaldehyde (4% in PBS) for 10 min at 4 °C. After fixation, the solution was replaced with buffer (0.1% Triton X-100, 4% paraformaldyhyde in PBS) for 3 min; then, the cells were washed three times with PBS and incubated with blocking buffer (5% bovine serum albumin and 5% normal goat serum in PBS) for 1 h at room temperature. The GF-1 cells were then treated with polyclonal rat anti-NNVCP antibody (1:200 in blocking buffer) and rabbit anti-Ckba (1∶500 in blocking buffer) for 2 h at room temperature. Next, the cells were washed three times with PBST (0.2% Tween-20 in PBS) and reacted with Cy3-conjugated goat anti-rabbit IgG antibodies (1∶1000 in PBS; Jackson ImmunoResearch) and anti-rat antibody (1∶1000 in PBS; Alexa flour 488) at room temperature. Counterstaining of the nucleus was performed with DAPI. After being washed three times with PBST, the cover glasses were wet mounted and the fluorescent signals were examined with a Leica TCS SP5 Confocal Spectral Microscope Imaging System.

Samples were relaxed 10 min in cold (4°C) relaxant solution (10 mM MgCl₂/5 mM NaCl/1 mM KCl/8% ethanol), fixed 30 min in 4% formaldehyde in 0.75x PBS, and rinsed in 1x PBS. Then they were permeabilized with 0.1% Triton-X in PBS (PBTx), blocked 1 h in 10% normal goat serum (NGS) in PBTx, and incubated 15–20 h at 4°C with mouse anti-acetylated-α-tubulin monoclonal antibody (T6793, Sigma, St. Louis, MO, USA) and rabbit anti-serotonin polyclonal antibodies (S5545, Sigma), both diluted 1:100 in blocking solution. Specimens were then washed in PBTx and incubated 15–20 h at 4°C in blocking solution containing Alexa-Fluor-647-conjugated goat anti-mouse IgG(H + L) antibodies (1:200, A21236, Invitrogen, Carlsbad, CA, USA) and Cy3-conjugated goat anti-rabbit IgG antibodies (1:200, 111-166-003, Jackson Immunoresearch,West Grove, PA, USA), 60 nM Alexa-Fluor-488 phalloidin (A12379, Invitrogen), and 10 μg/mL DAPI. After washing with PBTx and PBS, specimens were transferred through a graded glycerol series and mounted in 25 mM n-propyl-gallate in 75% glycerol/25% PBS.