Duolink in situ detection reagent kit

The Duo-Link II in situ proximity ligation assay (PLA) was carried out with the Duolink in situ detection reagent kit (Sigma-Aldrich) according to the manufacturer’s instructions, as described in our recent studies [37 (link),32 (link)]. Briefly, coronal parallel series of sections prepared as described above were blocked in 10% donkey serum for 1 h and followed by incubation with mouse and rabbit primary antibodies overnight at 4 °C. After washing, Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity secondary probes were added to the sections and incubated for 1 h at 37°C. Sections were washed twice, ligation mix was added to the samples and Incubated for 30 min at 37°C for ligation and circularization. Amplification and detection through rolling circle amplification was conducted after washing the sections according to the manufacturer’s protocol. Sections were then mounted on the slides, sealed in mounting medium with DAPI and the PLA signals as distinct dots were visualized and captured under a LSM700 confocal microscope at 40x magnification. The red spots representing protein-protein interaction were separated and analyzed using NIH Image J software.

Cells cultured in 8-well slide chamber were fixed and blocked in 5%
(vol/vol) donkey serum for 1 hour at room temperature and incubated overnight
with the respective target primary antibodies at 4°C. The cells were then
incubated for 1 hour at 37°C with the following Duolink proximity
ligation assayprobes: anti-rabbit MINUS (Sigma-Aldrich, DUO92005) and anti-mouse
PLUS (Sigma-Aldrich, DUO92003). Duolink in situ detection reagent kit
(Sigma-Aldrich, DUO92008) was used for ligation and amplification at 37°C
according to the manufacturer’s protocol. All images were acquired using
Leica WLL SP8 confocal laser scanning microscope with LASX image processing
interface.

Duo-Link II in situ proximity ligation assay (PLA) immunoassay was performed as described in the recent studies (Sareddy et al., 2015 (link); Tu et al., 2015 (link)). In brief, hippocampal tissue sections were blocked in 10% donkey serum for 1 hour and incubated overnight with appropriate primary antibodies at 4 °C. The slides were then incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes for 1 hour at 37 °C. Ligation and amplification were carried out using the Duolink in situ detection reagent kit (Sigma–Aldrich) according to the manufacturer’s protocol. Images were captured under LSM510 confocal microscope, and red spots representing the interactions were quantitatively analyzed using NIH ImageJ software.