Duolink in situ fluorescence kit

A549 and NCI-H460 cells were seeded in six-well plates with slides (1.5 × 10⁵ cells/well). Cells were transfected with SFB-Vector or SFB-GOLPH3 and fixed with 4% paraformaldehyde (PFA) for 10 min. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 for 3 min prior to blocking with 2% BSA (in PBS) for 0.5–1 h. Cells then were incubated with an appropriate concentration of primary antibody (mouse anti-FLAG: 1:100, Sigma, St. Louis, MO, USA; Cat. No. F1804; rabbit anti-CKAP4: 1:50, Novus, Colorado, USA; Cat. No. NBP1-26643; mouse anti-GOLPH3: 1:50, Proteintech, Rosemont, IL, USA; Cat. No. 67777-1-Ig) overnight at 4 °C. The PLA was performed using a Duolink in situ fluorescence kit (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. DUO92101) according to the manufacturer’s instructions. Images were acquired using DragonFly confocal imaging system (Andor) and data were analyzed using Image J and GraphPad Prism.

PLA was adapted from Rush et al., 2020 (link). Briefly, neurons on coverslips were fixed and permeabilized as with ICC, then were incubated overnight with primary antibodies for BIN1 (Santa Cruz, sc-30099, 1:500) and LVGCC-β1 (Abcam, S7-18, 1:1,000) overnight at 4°C, then PLA was performed using the Duolink In Situ Fluorescence kit (Sigma, DUO92004-100RXN). After PLA, coverslips were incubated with secondary antibody to view BIN1 and mounted with Duolink In Situ Mounting Medium with DAPI. Fluorescent images were taken using an epifluorescence microscope at 60x with four channels: DAPI (nuclei), FITC (PLA), and TRITC (mKate2). 7–9 images per slide were obtained and analyzed using ImageJ (v. 2.0.0-rc-69/1.52 p). PLA puncta were quantified using ImageJ particle analyzer, and the average number of puncta per field of view (FOV) or each coverslip was used for analysis.

PLA was carried out using Duolink in situ fluorescence kit (#DUO92101 from Sigma) according to the manufacturer's protocol. In brief, HeLa cells were simultaneously transfected with Myc-p85 plus control siRNA or AK0 siRNA in culture chambers and 48 h later, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X, followed by 3% BSA for blockage. Myc tag (mouse) and DHX9 (Rabbit) antibody were added and incubated at 4 °C overnight. Secondary antibodies conjugated with oligonucleotides (Rabbit antibody with PLA probe plus and Mouse antibody with PLA probe minus) were incubated for 1 h at 37 °C after primary antibody. After wash, ligation was taken place for 30 min at 37 °C, followed by amplification with polymerase for 2 h at 37 °C.

PLA was carried out using Duolink in situ fluorescence kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, HEK293 cells were transfected with pcDNA3.1-EphA2. Forty-eight hours later, cells were incubated with Flag-Wnt1 conditional medium (CM) for an additional 3 h, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking for blockage. Anti-Flag (mouse) and anti-EphA2 (Rabbit) antibodies were added and incubated at 4℃ overnight. Secondary antibodies conjugated with oligonucleotides (Rabbit antibody with PLA probe plus and Mouse antibody with PLA probe minus) were incubated for 1 h at 37 °C after primary antibody. After wash, ligation was taken place for 30 min at 37 °C, followed by amplification with polymerase for 100 min at 37 °C.

For proximity ligation assay (PLA), cells were grown overnight on 18-mm coverslips, fixed, permeabilized, and blocked as recommended by the manufacturer (DuoLink In Situ Fluorescence kit, Sigma-Aldrich). Cells then were incubated with mouse anti-ribophorin II and rabbit anti-LC3B (both diluted 1:200) overnight at 4°C. The cells were washed, treated for 1 h with the DuoLink in situ PLA probes, anti-mouse MINUS, and anti-rabbit PLUS, washed again, and incubated with ligase-containing ligation buffer for 30 min at 37°C. For amplification, the reactions were treated with polymerase in amplification buffer and incubated for 100 min at 37°C. The cells were washed, incubated with DuoLink in situ mounting medium containing DAPI (to stain nuclei) for 15 min, and subjected to analysis. The images of in situ PLA were viewed and captured using an IX71 fluorescence microscope. The same analytic parameters were used consistently throughout all experiments. The images shown are representative of at least three independent experiments carried out under the same conditions.