Duolink 2 fluorescence kit

The in situ proximity ligation assay (PLA) allows the visualization and subcellular localization of protein–protein interactions in individual fixed cultured cells, using secondary antibodies with attached oligonucleotides. When a pair of antibodies binds in proximity, the attached oligonucleotides can guide the creation of a DNA circle by ligation. This circle then templates a local rolling circle amplification reaction, whose product is easily detectable by FISH [27] (link). The HIF-1α interaction with ARNT under hypoxia was monitored in HeLa cells grown on slides. After appropriate incubation, cells were fixed with 3% formaldehyde in PBS for 5 min, permeabilized with PBS/Triton 1% for 15 min at 4 °C, incubated with anti-HIF-1α and/or anti-ARNT antibodies for 16 h at 4 °C and processed using the Duolink II Fluorescence Kit (Olink Bioscience). Slides were counterstained with DAPI (100 μg/ml) before mounting. Images of in situ PLA experiments were taken in a Zeiss Axioplan fluorescence microscope using an AxioCam MRm CCD sensor and 40 × objective with filters for DAPI, FITC and Cy3. PLA signals were digitally quantified using the ITCN tool of public domain software for image analysis ImageJ [28] (link).

PLA assay was performed in cells cultured on glass coverslips and on gastric carcinoma tissue sections. According to previous studies, PLA is an appropriate approach for protein-glycan interaction detection in tissues 31 (link). Thus, we used this approach to evaluate the presence of SLe^x on CEA in tissue sections. Briefly, paraffin tissue sections were dewaxed and rehydrated followed by antigen retrieval with citrate buffer. Tissue sections and cells on glass coverslips were then incubated with normal goat serum diluted 1:5 in 10% BSA PBS followed by incubation with a solution containing the two monoclonal antibodies overnight at 4 °C. PLA probes anti-IgG plus and anti-IgM minus were incubated 1 h at 37 °C. PLA reactions were performed using the DuoLink® II Fluorescence Kit (Olink® Bioscience) according to the manufacturer's instructions. Nuclei were stained with DAPI and slides were mounted in an appropriated medium (Duolink II). PLA products were seen as fluorescent red dots. Fluorescence was examined in a fluorescence microscopy and images were acquired using a Zeiss Axio cam MRm and the AxioVision Rel. 4.8 software (Carl ZEISS).