Duolink in situ detection reagents red kit

Cells were seeded in VitroGel at 50% confluence on chamber slides for PLA (Proximity Ligation Assay) using the Duolink in situ Detection Reagents Red kit (DUO92008, Sigma-Aldrich), following the given protocol. Various groupings of primary antibodies targeting DCLK1 and KIF16B, KIF1A, KIF1B, KIF1C, KIF26B, MMP1, MMP9, or RAB40B were employed. Subsequently, PLA puncta were imaged utilizing a TCS SP8 confocal laser scanning microscope (Leica Microsystems) equipped with a 40×1.35 NA or 100×1.35 NA oil immersion objective. High-resolution z-stack photograms were captured with a z-interval of 0.5 μm. Two-dimensional projections were generated using the LAX software platform (Leica Microsystems) by combining the maximum intensity from each z-series. The density of PLA puncta was determined by analyzing high-resolution images (captured with a 40×1.35 NA objective) using ImageJ (NIH).

Contact between ER and lysosome was studied using a Duolink InSitu Detection Reagents Red kit (Sigma) according to the manufacturer’s protocol. Briefly, cells were incubated with primary Abs against ORP1L (Abcam, 1:200) and VAPA (Santa Cruz, 1:200) at 4 °C overnight. After washing, cells were incubated with PLA plus and minus probes at 37 °C for 1 h. After ligation reaction to close the circle and rolling circle amplification (RCA) of the ligation product, fluorescence-labeled oligonucleotides hybridized to the RCA products were observed by fluorescence microscopy.

Interactions between NEU1 and ALK5 was detected by in situ proximity ligation assay (PLA) in HK-2 cells and human kidney biopsy sample. For HK-2 cells, cells were grown on glass bottom dish and fixed with 4% paraformaldehyde in PBS for 15 min. For paraffin-embedded human kidney biopsy sample, following dewaxing and rehydration of tissue sections, antigen retrieval was performed by heating the slides for 30 min at 95 °C in Tris-EDTA buffer. From this point, the tissue sections and the fixed HK-2 cells were treated identically, and the PLA protocol was followed according to the manufacturers’ instructions. The In-situ PLA (Duolink® in situ Detection Reagents Red Kit, Sigma-Aldrich, DUO92008; Duolink® in situ PLA Probe Anti-Rabbit PLUS, Sigma-Aldrich, DUO92002; Duolink® in situ PLA Probe Anti-Mouse MINUS, Sigma-Aldrich, DUO92004) was performed according to manufacturer’s instruction. Fluorescence signal amplification was observed using the Zeiss LSM800 confocal laser scanning microscopy.

MCF-7 cells were cultured for 24 h on coverslips and then fixed and permeabilized using cold methanol for 20 min at −20°C. P-LISA staining was performed according to the OlinkBioscience's recommendations using Duolink® In Situ Detection Reagents Red kit (Sigma-Aldrich, DUO92008) and as previously described [51 (link)]. The number of red dots and the intensity per dots were counted using the Blobfinder software. 200 cells were randomly selected in 5 fields.

PLA was performed using the Duolink In Situ Detection Reagents Red Kit (DUO92008, Sigma-Aldrich), according to the manufacturer's protocol. Briefly, HEK 293T ΔN1-3 V5-NOTCH3 and HEK 293T ΔN1-3 V5-NOTCH3^L1519P cells were cultured for 24 h on coverslips and fixed with 4% paraformaldehyde in PBS for 10 min, followed by blocking with 5% bovine serum albumin (BSA) in 0.1% Triton X-100 for 1 h at 37°C. Primary antibodies, anti-1E4 (mouse, Sigma-Aldrich) and anti-NOTCH3 (rabbit, Abcam), were incubated overnight at 4°C (see Table S3 for antibody dilutions). After washes with PBS, the slides were incubated with the PLA probes anti-mouse MINUS (DUO92004, Sigma-Aldrich) and anti-rabbit PLUS (DUO92002, Sigma-Aldrich), according to the manufacturer's protocol. The plasma membrane was stained using the anti-Na/K ATPase-Plasma Membrane Marker (Alexa Fluor^® 488) (Abcam). Subsequently, the coverslips were washed with PBS and mounted with Duolink in situ mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI).