Anti mouse plus

Duolink Proximity Ligation Assay³⁸ was used to study interaction between Rab7 and RILP. Briefly the primary neurons from WT and Cln1^−/− mice were grown in chamber slides and fixed with methanol for 15 to 20 min at −20°C. The cells were blocked with goat serum and incubated with Rab7 antibody (Abcam; Cat#ab50533; dilution 1:1000) and RILP antibody (Abcam; Cat#ab140188; dilution 1: 3000) overnight at 4°C. After three washes with PBS the cells were incubated with anti-rabbit-MINUS (Sigma–Aldrich; Cat#DUO92005 Anti-RILP) and anti-mouse–PLUS (Sigma–Aldrich; Cat#DUO92001-for Rab7 antibody) PLA probes and subjected to ligation and amplification reaction using Duolink® In Situ Detection Reagents Green (Sigma–Aldrich; DUO9201) according to manufacturer’s protocol. For controls, cells were similarly processed but without primary antibody or with one primary antibody only. When the primary antibodies were in close proximity (<40 nm apart) fluorescent signals were detected as green dots under the FITC channel. The cells were mounted with DAPI-Fluoromount G (Thermo Fisher, Cat#010020) and visualized using an LSM710 confocal microscope. High resolution z stack images were captured at 40X, the z stacks were merged by maximum intensity projection using Zen Desk software to include all the PLA signals and quantitated using Duolink image tool.

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.

Protein interactions between GHSR/DRD1 and GHSR/β-arrestin 2 in mouse brain slices and hippocampal neuron cultures were detected using Duolink Proximity Ligation Assay (PLA) detection kits (Sigma-Aldrich, #DUO92008) following manufacturer’s instructions. The following primary antibodies were used in proper combinations: goat-anti-GHSR (Santa Cruz Biotechnology, #sc-10359, 1 : 100), rabbit-anti-DRD1 (Abcam, #ab81296, 1 : 200), mouse-anti -arrestin 2 (Santa Cruz, #sc-13140). The specificity of antibodies to GHSR and DRD1 was validated as previously described [3 (link)]. The following Duolink in Situ PLA Probes were used: anti-Rabbit PLUS (Sigma-Aldrich, #DUO92002), anti-Goat MINUS (Sigma-Aldrich, #DUO92006), anti-Mouse PLUS (Sigma-Aldrich, #DUO92001). Images were collected on a Nikon confocal microscope. PLA-positive dot number was counted using Nikon-Elements Advanced Research software.