Proximity Ligation Assay for DRG Proteins

PLA was performed using PLA kits (Sigma) as previously described (19 (link)). Antibodies used were ANO1 (1:200; Santa Cruz or 1:500; Abcam), TRPV1 (1:500; Neuromics) and IP₃R1 (1:500; Calbiochem), CD71 (1:500, Santa Cruz). For anti-guinea pig antibodies (such as TRPV1), PLA ‘minus’ probes were manually conjugated onto an anti-guinea pig IgG antibody using a PLA conjugation kit. DRG cultures were prepared and plated on microscope slides (coated with poly-d-lysine and laminin) and permeabilized using acetone:methanol (1:1) on ice for 20 minutes and washed with PBS 3 times. Hydrophobic barriers were made on the slides using an ImmEdge hydrophobic barrier pen (Vector laboratories) to delimit reactions to ~1cm² and blocking was done using the PLA blocking reagent for 30 minutes in a humidified incubator (37°C). Primary antibodies were then applied and left at 4°C overnight. The following day, PLA probes were applied to the samples; signals were amplified and detected according to the manufacturer’s instructions; and slides were sealed with DAPI-containing mounting medium (Sigma). Samples were imaged using the Zeiss LSM700 confocal microscope. Green fluorescent puncta (0.5–1μm) were counted per cell using Zen imaging software (Zeiss).