Alexa fluor 488 568 633 igg

48 h post transfection with dPIP5K^L::GFP, cells were fixed in 2.5% PFA for 20 min. Upon fixation, cells were washed thrice with PBTx (1× PBS + 0.3% Triton X-100) for 10 min each, permeabilized with 0.37% Igepal (Sigma-Aldrich) in 1× M1 for 13 min and then blocked for 1 h at room temperature in 1× M1 containing 5% FBS and 2 mg/ml BSA. Post blocking, the cells were incubated with primary antibodies in blocking solution overnight at 4°C. The following antibodies were used: anti-GFP (1:5,000; Abcam [ab13970]) and anti-PNUT (1:250; DSHB [4C9H4]). Next morning, the cells were washed thoroughly, and incubated with the appropriate secondary antibodies conjugated with fluorophore at 1:300 dilutions (Alexa Fluor 488/568/633 IgG, [Molecular Probes]) and incubated at room temperature for 2 h. The cells were then washed and imaged on an Olympus FV3000 confocal microscope using Plan-Apochromat 60×, NA 1.4 objective (Olympus). Image analysis was performed using ImageJ from NIH.

S2R⁺ cells stably expressing Actin-GAL4 were transfected using Effectene based as a transfection agent (Effectene Qiagen Kit [301425]). S2R⁺ cells were plated in 12-well plates; 0.5 μg of desired DNA construct was used for transfections. Forty-eight hours post transfection cells were allowed to adhere on glass cover-slip dishes for 1 h, fixed with 2.5% PFA in 1× M1 buffer for 20 min. They were washed and permeabilized with 0.37% Igepal (Sigma) in 1× M1 for 13 min. Blocking was performed in 1× M1 containing 5% FBS and 1 mg/ml BSA, for 1 h at RT. Then the cells were incubated with primary antibodies diluted in block solution for 2 h at RT, washed with 1× M1 several times, and incubated with appropriate secondary antibodies for 1–2 h at RT. These were then washed and imaged on an Olympus FV1000/FV3000 confocal microscope. The following antibodies were used: chicken anti-GFP(1:5000, Abcam [ab13970]), rabbit anti-hPLD1^N229 (1:200, kind gift from Dr Nicholas Ktistakis), and rabbit anti-MYC (1:400, CST [2272S]). Appropriate secondary antibodies conjugated with a fluorophore were used at 1:300 dilutions [Alexa Fluor 488/568/633 IgG, (Molecular Probes)]. Alexa Fluor 488-phalloidin (1:400, Invitrogen [A12379]) was used to stain the F-actin (plasma membrane).

For Immunohistochemistry, retinae from 1-d old flies were dissected in chilled PBS, followed by fixation in 4% paraformaldehyde in PBS with 1 mg/ml saponin at room temperature for 30 min on orbital shaker. Fixed retinae were washed three times in PBTx (1× PBS + 0.3% Triton X-100) for 10 min each. The sample was then blocked using 5% FBS in PBTx for 2 h at room temperature on shaker, after which the sample was incubated with primary antibody in blocking solution overnight at 4°C on a shaker. The following antibodies were used: anti-GFP (1:5,000; Abcam [ab13970]) and anti-HA (1:50, CST [6E2]). Appropriate secondary antibodies conjugated with a fluorophore were used at 1:300 dilutions (Alexa Fluor 488/568/633 IgG; [Molecular Probes]) and incubated for 4 h at room temperature. Wherever required, during the incubation with secondary antibody, Alexa Fluor 568-phalloidin (1:200; Invitrogen [A12380]) was also added to the tissues to stain the F-actin. After three washes in PBTx, samples were mounted in 70% glycerol in 1× PBS. Whole mounted preparations were imaged on Olympus FV3000 confocal microscope using Plan-Apochromat 60×, NA 1.4 objective (Olympus). Image analysis was performed using ImageJ from NIH.