Alexa fluor 488 anti mouse secondary antibody

Caco-2 cells grown on membrane inserts were washed in PBS, permeabilized for 2 min with PIPES/EGTA—saponin 0,25% (piperazine-N–N’-bis-(2-ethane sulphonic acid)/ ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N’-tetraacetic acid-saponin 0,25%), fixed for 20 min with 3% paraformaldehyde in PBS, and washed with PBS. The membrane inserts were cut in two pieces and blocked for 30 min with 3% Bovine Serum Albumin (BSA) (Sigma-Aldrich) and 0,3% Triton X-100 in PBS. For F-actin localization, cells were incubated for 20 min with Actin-Stain 555 Phalloidin (Cytoskeleton) and washed 3 times in PBS. E-cadherin was stained with Anti-E-Cadherin primary antibody (Santa Cruz Biotechnology, sc-21791) diluted 1:400 and, after 3 washes, with Anti-mouse AlexaFluor 488 secondary antibody (Invitrogen) diluted 1:400. Nuclei were labeled with 4’, 6-diamidino-2-phenylindole (DAPI) (1 µg/mL). Membranes were mounted with Mowiol 4–88 (Calbiochem) and viewed with the ZEISS LSM700 (Germany) confocal laser-scanning microscope with a 63 × /1.40 NA oil immersion objective.

Embryos were fixed overnight at room temperature in 4% paraformaldehyde in 0.1 M PO₄ buffer, then rinsed with PBS/0.1% Tween-20 (PBST) and blocked with incubation buffer (IB: 2 mg/mL BSA, 1% Sheep Serum, 1% DMSO, 0.5% Triton X-100 in PBS) for at least 1 hr. Embryos were incubated overnight at 4 °C with monoclonal anti-HNK1 antibody in IB (ZN-12, 1:250; Zebrafish International Resource Center), rinsed in PBST, then incubated with anti-mouse AlexaFluor 488 secondary antibody (1:1000; Invitrogen) for at least 2 hr at room temperature or overnight at 4 °C. For double labeling with anti-acetylated tubulin (T6793, 1:1000, Sigma), the IgG subtype-specific secondary antibodies anti-mouse IgG1 AlexaFluor 488 and anti-mouse IgG2b AlexaFluor 647 (1:1000, Invitrogen) were used.

Vero E6 cells were grown in coverslip bottom dishes of diameter, 35 mm (Biogenuix, New Delhi, India) and infected with SARS-CoV 2, Isolate USA-WA1/2020 virus using a multiplicity of infection (MOI) of 10. After 24-h post-infection (pi), the cells were fixed with 100% chilled methanol for 20 min in ice. In order to visualize the infected cells intracellularly by immunofluorescence, fixed cells were blocked with 3% goat sera for 1 h at RT followed by incubation with pooled sera (1:50 dilution of S1-pep1 and S2-pep3 and 1:100 dilution of RBD-pep2 pooled sera) from each immunized group and PBS-control sera (1:100 dilution) for 3 h at RT. The cells were then washed in PBS three times and stained with the anti-mouse AlexaFluor 488 secondary antibody (Invitrogen Corporation, CA, USA) at 1:1000 for 1 h at RT. The cells were mounted using ProLong Gold anti-fade reagent with DAPI (Invitrogen Corporation, CA, USA). Images were acquired on an Olympus FV3000 confocal microscope with 60X (NA 1.4) Plan Apo objectives.

DNA double-stranded breaks (DSBs) and blocked replication forks lead to the phosphorylation of histone protein H2AX on serine 139 (Muslimovic et al. 2008 (link)). We performed a γH2AX staining to investigate the DNA damage by the procedure as previously described by Mariotti et al. (2013 (link)) with some modifications. Briefly, after treatment with various pH and B[a]P at different time points, A549 cells were fixed in 2% formaldehyde for 20 min at room temperature. After fixation cells were permeabilized with 0.5% Triton X-100:PBS for 20 min at 4 °C and then blocked with 2% BSA and 0.5% Tween-20 in PBS. Cells were then stained with anti-γH2AX antibody (Upstate via Merck Millipore) overnight at 4 °C and the use anti-mouse Alexa fluor 488 secondary antibody (Life Technologies) for 1 h at 37 °C. Coverslips were mounted with VECTASHIELD^® Mounting Medium containing DAPI, to counterstain cellular nuclei. γH2AX stained cells were scored manually by a digital fluorescent microscope with 100X objective, and the average number of positive cells was calculated from a minimum of 100 cells per dose/time point. Experimental data present the average of 4 independent experiments.