Alexa fluor 633 goat anti mouse antibodies

MCF-7 and MDA-MB-231 cells non-infected or infected with HCMV MOI of 5 were fixed at 6 days post infection (dpi). Cells were treated in Fix &Perm Medium A and B according to the supplier`s instruction (Molecular Probes) and co-stained with primary antibodies HCMV IE (Merck) and COX-2 (CellSignaling) or 5-LO (Abcam) diluted in phosphate-buffered saline (PBS) containing 1% BSA (Sigma) for 45 min at 4 °C. After washing with PBS, cells were incubated with secondary Alexa Fluor 633 goat anti-mouse antibodies (Life Technologies) and Alexa Fluor 488 anti-rabbit antibodies (Invitrogen). Rabbit IgG (Biocare Medical) and mouse IgG2a (Dako) served as negative controls. Acquisition was performed using Flow cytometry (FACS) and data were analyzed by Summit 4.3 software. (Three separate experiments were performed).

Larvae were fixed in 4% paraformaldehyde/phosphate‐buffered saline (PBS), washed in PBS, dehydrated using methanol 100% and stored at −20°C overnight, then treated with 100% acetone in −20°C for 10 minutes. The rehydrated samples were blocked in blocking solution (PBS + 10% goat serum + 1% DMSO + 0.3% Triton X100) that was then replaced with fresh blocking solution with commercial primary antibodies. Acetylated tubulin antibodies (T6793; Sigma‐Aldrich; 1:1000) were used followed by Alexa Fluor 633 goat anti‐ mouse antibodies (A‐21052; Life Technologies; 1:200). For BTX + SV2 staining, samples were incubated with Alexa Fluor 488 conjugated α‐Bungarotoxin (B13422; Invitrogen; 1:300) overnight at 4°C, washed, incubated overnight at 4°C with synaptic vesicle antibodies (AB_2315387; DSHB; 1:100), washed and incubated overnight at 4°C with Alexa Fluor 633 goat anti‐mouse antibodies.