Fitc conjugated goat anti rabbit igg antibody

An immunofluorescence assay (IFA) was carried out to examine the susceptibility of different cell lines to pFPV-sc. Briefly, cells on 24-well plates were infected with the virus at a MOI of 0.1, then incubated at 37°C, 5% CO₂ prior to washing with PBS and fixation with 4% Paraformaldehyde. Cells were then blocked with PBS containing 2% bovine serum albumin (Sigma), and incubated with 200 μl rabbit hyperimmune serum at a 1:150 dilution. One hour later, cells were washed three times with PBS, followed by another incubation step with 200 μl FITC-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) at 1:400 dilution mixed with 4′, 6-diamidino-2-phenylindole (DAPI) at 1:1000 dilution. Lastly, cells were washed, mounted on glass slides and visualized under a fluorescence microscope.

Rabbit polyclonal anti-PDK1 antibody was purchased from Abcam (Cambridge, MA, United States; Cat#: ab92959); Rabbit polyclonal anti-PDHE1α (pSerine232); Rabbit polyclonal anti-PDHE1α (pSerine293) and Rabbit polyclonal anti-PDHE1α (pSerine300) antibodies were purchased from EMD Chemicals (Beverly, MA, United States; Cat#: AP1063, AP1062 and AP1064); Myc-Tag Rabbit monoclonal antibody (cell signaling technology; Cat#:2278); Mouse monoclonal anti-β-actin and anti-α-tubulin-FITC antibodies were purchased from Sigma (St. Louis, MO, United States; Cat#: F2168 and A5441); FITC-conjugated goat anti-rabbit IgG antibody was purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Human dental pulp cells fixed in 4% paraformaldehyde (PFA; T&I, BFA-9020) and deparaffinized tissue sections were treated with phosphate-buffered saline (PBS) containing 0.5% Triton X-100 (Amresco, 0694) for permeabilization. After being washed and blocked, the cells were incubated for 1 h with LC3B antibody (1:100) or α-TUBULIN (1:100, YOL1/34; Santa Cruz Biotechnology, sc-53030) antibody and TAU antibody (1:50) in blocking buffer (PBS and 2% bovine serum albumin (BSA; Gibco BRL, 30063-572)). Tissue sections were incubated with α-TUBULIN antibody (1:200) overnight at 4°C. Subsequently, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (1:200; Thermo Fisher Scientific, F2765), Alexa Fluor 488-conjugated goat anti-rat IgG antibody (1:200; Thermo Fisher Scientific, A11006), or Cy3-conjugated goat anti-mouse IgG antibody (1:200; Merck Millipore, AP124C) was applied. After being washed, the chromosomal DNA in the nucleus was stained with 4’,6-diamidino-2-phenylindole (DAPI) during the mounting procedure (Vector Labs, H-5000). Samples were visualized using confocal laser scanning microscopy (Carl Zeiss, LSM 800).