Fitc labeled goat anti mouse igg

Verification of virus recovery was performed using immunofluorescence on Vero cells grown in chamber slides. A small (~ 100ul) of the transfection supernatant was transferred onto the Vero cells. After incubation for 4 days at 37°C, the media in the chamber was removed and the monolayer was washed with PBS and then fixed with 80% acetone. The fixed cells were then incubated at 37⁰ C with 1:1000 dilution of a pan-flavivirus monoclonal antibody, 4G2 [22 (link)] for 1 h. The cells were then washed and incubated with 1:1000 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Life Technologies) for 45 min. Finally, the monolayers were washed and covered with a coverslip and observed under a fluorescent microscope (Zeiss).

Protein expression was measured using flow cytometry analysis as previously described [39 (link)]. Briefly, HEK293 cells were transfected with either RABV-G mRNA or Luc mRNA (negative control) for 24 h. Cells were then collected and stained with a monoclonal mouse anti-rabies antibody (HyTest Ltd, Turku, Finland) and an FITC-labeled goat anti-mouse IgG (Life Technologies GmbH, Darmstadt, Germany). Flow cytometric analysis of FITC-positive cells confirmed protein expression.

The dissected brain tissues were immediately immersed in 4% PFA for 24 h and embedded in paraffin after a sequence of dehydration processing. The dewaxing processes were then carried out after the paraffin blocks were divided into 4 μm thick slides. The brain sections were then rinsed three times in PBS and placed in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. The sections were treated with a primary antibody overnight at 4 °C after being blocked with 10% Bovine Serum Albumin (BSA) (ST023, BEYOTIME, Shanghai, China) for an hour. Primary antibody: anti-tyrosine hydroxylase (TH, 1:200, Abcam, Cambridge, UK) and anti-ionized calcium-binding adapter molecule 1 (IBA-1, 1:100, CST, USA). After half an hour of rewarming, appropriate secondary antibody FITC-labeled goat anti-mouse IgG (1:500, Thermo Fisher, Waltham, MA, USA) was used to detect the primary antibody for 1 h, which was observed under the fluorescence microscope (Nikon eclipse 80 i; Nikon, Tokyo, Japan). The results of each group were analyzed by Image J software, version, 1.53 (NIH, Bethesda, MD, USA).

RD cells were seeded in 6-well cell culture plates. The cells were infected with 7 strains of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of Enterovirus A, 1 strain of Echovirus 11 of Enterovirus B, and 1 strain of poliovirus Sabin 3 of Enterovirus C for 24–48 h. The infected cell monolayers were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.1% Triton-X 100 in PBS for 10 min at room temperature. Cells were then washed with PBS and blocked with PBS containing 1% bovine serum albumin (BSA) at 37 °C for 30 min before being incubated with 1 μg /mL mAb 1A11 for 1 h at 37 °C. Cells were rinsed three times with PBS and incubated with 0.2 μg/mL of FITC-labeled goat anti-mouse IgG (Thermo Fisher, Waltham, MA, USA) for 1 h at 37 °C. The cells were rinsed again in PBS and examined under an inverted fluorescence microscope (Leica, Wetzlar, Germany).

Immunofluorescent cytochemical staining for γoH2AX foci was performed by exposing cells grown on coverslips to LB100 (2.5 μM) for 3 hours prior to administration of 8 Gy or sham radiation. Cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.5% Triton X-100 in saline, blocked with 15% FBS in PBS, and incubated in blocking buffer containing primary antibody against γ-H2AX (Millipore) and then incubated with FITC-labeled goat anti-mouse IgG (Invitrogen, Carlsbad, CA). Nuclei were counterstained with DAPI (Sigma, St. Louis, MO). Cover slips were mounted with Beyotime anti-fade solution (Beyotime institute, Jiangsu, China) and γ-H2AX foci were imaged (40x objective) with a fluorescent microscope (BX51 Olympus microscope, Tokyo, Japan) and a Evolution^TM VF camera (Media Cybernetics, Rockville, MD).

Immunofluorescence staining was performed to determine the nuclear distribution of p-ATM foci in H460 and A549 cells using image analysis. Cells were grown on chambered slides 1 day prior to irradiation or digoxin treatments. After digoxin (40 nM) exposure for 24 h, cells were irradiated and incubated for 1 or 24 h before harvest. Cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.6% Triton X-100 in PBS, blocked with 4% FBS in PBS, and incubated in blocking buffer containing primary antibody against p-ATM (Santa Cruz Biotechnology, San Diego, CA, U.S.A.) and then incubated with FITC-labeled goat anti-mouse IgG (Invitrogen, Carlsbad, CA). Nuclei were counterstained with DAPI (Sigma, St. Louis, MO). Coverslips were mounted with fluorescence mounting medium. The slides were examined using a fluorescence microscope with digital imaging system (Olympus, Tokyo, Japan) and images were captured with a charge-coupled device camera. For quantitative analysis, foci-positive cells were counted in at least 50 cells from randomly captured images.

P. berghei asexual stages, gametocytes and subsequent developmental stages (gametes, zygotes and ookinetes) were fixed with 4 % paraformaldehyde in PBS for 15 min at room temperature, rinsed with 50 mM glycine in PBS, and blocked with PBS containing 5 % skimmed milk for 60 min at 37 °C. Slides were incubated with mouse anti-PbPH sera (1:200) or anti-Pbs21 mAb (clone 13.1, 1:500) at 37 °C for 60 min, washed 3 times in PBS, and then incubated with FITC-labeled goat anti-mouse IgG (1:500, Invitrogen) at 37 °C for 30 min. Parasite nuclei were counter-stained with 1 μg/mL of 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen) [20 (link)]. Slides were then mounted with ProLong® Gold anti-fade reagent (Invitrogen) and visualized by fluorescence microscopy. Images were captured and processed on a Zeiss Axio Observer Z2 using Axiovision software and Adobe Photoshop.