Anti mouse igg antibody conjugated to fitc

HEK293 cells were grown to 70% confluence on coverslips in six-well plates before fixation with 4% paraformaldehyde in PBS for 15 min. Following fixation, cells were permeabilised with 0.1% Triton X-100 in PBS for 5 min and then blocked in 250 μl 3% BSA in PBS for 15 min at room temperature. 25 μl droplets of anti γ-tubulin (1:1000, Sigma, GTU88) primary antibody were applied to a sheet of parafilm and the coverslips were placed cell-side down on the drops and left in a moist environment overnight at 4°C. Coverslips were then placed on four 100 μl droplets of PBS with 0.1% Tween 20 sequentially and left on each for 5 min to wash. Coverslips were incubated with anti-mouse-IgG antibody conjugated to FITC (1:1000, Sigma) for 1 h at room temperature before being washed a further four times on 100 μl droplets of PBS with 0.1% Tween 20 and then placed cell-side down onto 25 μl droplets of DAPI (1 µg/ml) for 1 min followed by two 10 min washes with PBS. 100 μl 0.1% p-Phenylenediamine anti-fade (Sigma P6001-50G) was mixed with 900 μl 10% Mowiol mounting solution (Sigma 81381-50G), 5 μl droplets of this mounting mix were added to glass slides and the coverslips were placed, cell-side down, on top of the droplets before visualisation.

Cells were grown to the desired phase of growth and fixed in 5% formaldehyde for 1 h. Cells were washed twice in sorbitol buffer (1.2 M sorbitol, 0.1 M potassium phosphate buffer pH7.5), re-suspended in 0.5 ml sorbitol buffer plus 1 μl β-mercaptoethanol and 20 μl 1 mg/ml zymolyase, and incubated at 37°C for 40 min. Cells were applied to poly-L-lysine-coated slides with wells and 10 μl of 0.1% SDS added for 30 s before washing ten times with PBS plus 1 mg/ml BSA. Immobilised cells were incubated with primary monoclonal anti-β-tubulin antibody (1:500 dilution, Sigma, clone AA2) overnight at 4°C. Slides were washed a further ten times with PBS with 1 mg/ml BSA before adding 15 μl of the secondary anti-mouse-IgG antibody conjugated to FITC (1:1000, Sigma) in PBS with BSA at room temperature for 1 h. Slides were washed ten more times and then a drop of phenylenediamine mounting solution containing DAPI at 1 mg/ml was added before visualisation.