Cy3 conjugated goat anti mouse igg2a

Anti-CD31 (1:500, M0834), anti-CD68 (1:50, M0876), anti-CD3 (1:50, M0742), anti-CD20 (1:50, CD20cy), and fluorescein isothiocyanate (FITC)-conjugated anti-fibrin (1:100, F0111) primary antibodies were purchased from Dako (Glostrup, Denmark); anti-SMA (1:1000, A5228) and β-tubulin III (1:1000, T5076) were obtained from Sigma-Aldrich (St. Louis, MO, USA); anti-collagen IV (1:50, 2150-0140) and anti-CD40 (1:100, MCA 1590) were purchased from AbD Serotec (Oxford, UK); anti-pERK1/2 (1:100, #9101) and anti-PTEN (1:500, #9559) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-HIF-1α (1:100, sc-10790) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-MK2 (1:200, ADI-KAP-MA015) was obtained from Stressgen (Victoria, Canada). Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG1a, Cy3-conjugated goat anti-mouse IgG2a and Alexa Fluor 647-conjugated donkey anti-rabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Coverslips were coated with Poly-l-Lysine 0.01% for 10 min at room temperature and dried for 2 h. Cells were incubated for 30 min at 37 °C to let them adhere to the coverslip. Medium was removed and cells were fixed with PFA 4% for 10 min. Cells were incubated for 1 h with the following primary antibodies diluted in PBS/BSA 1%/Saponin 0.05%: mouse IgG1 monoclonal anti-Gag (clone KC57; Beckman-Coulter; dilution 1:75) and mouse IgG2a monoclonal anti-p17 (NIBSC CFAR; Cat#0342; dilution 1:100). After three washes, cells were incubated for 1 h with the following secondary antibodies diluted in PBS/BSA 1%/Saponin 0.05%: Alexa Fluor 488-conjugated rabbit anti-FITC (Invitrogen; dilution 1:100) and Cy3-conjugated goat anti-mouse IgG2a (Jackson ImmunoResearch; dilution 1:500). Coverslips were washed with PBS and water and mounted in DAPI-containing mounting medium (SouthernBiotech). Images were acquired on a LSM 700 confocal microscope (Zeiss) using ZEN software (ZEISS). Images were analyzed with Fiji software. Briefly, regions of interests (ROI) were drawn manually around each infected cell. Intensities of total Gag-Alexa Fluor 488 and p17-Cy3 signals were calculated in each ROI. Between 49 and 138 cells were analyzed per condition.