Pla probe anti mouse minus

Cells were fixed in 1× PBS, 4% paraformaldehyde for 20 min and permeabilized for 10 min with 0.4% Triton X-100, 0.05% CHAPS. The EBNA1–digoxigenin probe mRNA (5′-CTTTCCAAACCACCCTCCTTTTTTGCGCCTGCCTCCATCAAAAA-3′) at 50 ng/well was denaturated for 5 min at 80°C. The probe hybridization reaction was carried out in 40 μl of hybridization buffer (10% formamide, 2× SCC, 0.2 mg/ml E. coli tRNA, 0.2 mg/ml salmon sperm DNA and 2 mg/ml BSA). The fixed cells were washed and blocked into the blocking solution (1× PBS, 3% BSA, 0.1% saponin) before incubation with the primary antibodies (anti-digoxigenin, Sigma 1/200 and anti-NCL, Abcam 1/1000). The PLA reaction was performed under the manufacturer’s protocol using the Duolink PLA in situ kit, PLA probe anti-rabbit plus, PLA probe anti-mouse Minus and the in situ detection reagent FarRed, all from Sigma. The results were analysed using a Zeiss Axio Imager M2. All the PLA experiments were performed at least three times independently, and the following controls probes were implemented: without mRNA probe or without primary antibodies.

PLA was performed using Duolink In Situ PLA Reagents Red (Sigma-Aldrich, Cat#DUO92008) according to the manufacturer’s protocol. Ramos cells were attached to coverslips with 0.1% poly-L-lysine (Sigma-Aldrich, Cat#P8920) for 1 h at RT. Cells were permeabilized, fixed with methanol for 10 min at −20 °C, blocked with Duolink blocking solution, and incubated with primary antibodies. PLA Probe Anti-Mouse MINUS (Sigma-Aldrich) and PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich) were used as secondary probes. Samples were mounted with DAPI. PLA signals were detected using a Nikon Ti Microscope (λex = 594 nm; λem = 624 nm). Images were taken with a Nikon DS-Qi2 camera. PLA foci were counted automatically with ImageJ [57 (link)] after threshold adjustment using function “analyze particles”.