Alexa fluor 594 conjugated donkey anti mouse secondary antibody

BV2 cells were treated with LPS in the presence or absence of TCA (10 μM) for 24 h in 24-well plate followed by three times with PBS. The medium was removed and cells were fixed with ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 min. Cells were permeabilized with 0.2% Triton X-100 for 10 min followed by blocking with 10% normal donkey serum (Jackson ImmunoResearch Lab, West Grove, PA, USA) for 30 min at room temperature. Fixed cells were incubated with anti-CD11b monoclonal antibody (1 : 500) in PBS containing 0.1% Triton X-100 overnight at 4°C and then Alexa Fluor 594-conjugated donkey anti-mouse secondary antibody (1 : 500; Invitrogen) for 1 h at room temperature. The nuclei were counterstained using DAPI. Images of microglia were visualized under an Axiovert 40 CFT visible/fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Breast Cell lines were cultured in 8 well chamber slides, fixed, permeabilized and stained with anti-a2V (2C1) (mouse monoclonal antibody to a2V), was generated as previously described [34 (link)] specific for aa 488–510, the transmembrane region of the protein. Alexa Fluor® 594-conjugated donkey anti-mouse secondary antibody (1:200 dilution) (Invitrogen) was used. The cells were prepared for viewing using ProLong® Gold (Invitrogen) mounting medium containing DAPI. Stained cell lines were imaged by an Olympus Fluoview FV10i confocal microscope and analyzed by FV10i Fluoview Ver.3.0 software.