In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

Antibodies

Primary antibodies

The phosphorylation state-specific rabbit MOP antibodies pT370-MOP (7TM0319B), pS375-MOP (7TM0319C), pT376-MOP (7TM0319D), pT379-MOP (7TM0319E), the phosphorylation state-specific rabbit CB1 antibody pS425-CB1 (7TM0056A) as well as the phosphorylation-independent rabbit antibody np-MOP (7TM0319N) and phosphorylation-independent guinea pig antibody np-MOP (7TM0319N-GP) were provided by 7TM Antibodies (www.7tmantibodies.com). In addition, we used rabbit np-MOP from abcam (ab134054), rabbit np-CB1 from Cayman (1000659) and goat np-CB1 from Frontier Institute (MFSR100610). Additional information in Supplemental Table 1.

Secondary antibodies

We used a biotinylated AB from Jackson Immuno Research (711-065-152, anti-rabbit) and the respective streptavidine conjugates from ThermoFisher (S21381, Alexa FluorTM 555 conjugate) and (S11223, Alexa FluorTM 488 conjugate) for the amplified staining procedure. For the direct stainings we used CyTM3 (706-165-148, anti-guinea pig) and CyTM3 (705-165-147, anti-goat), both obtained from Jackson ImmunoResearch.

Animals

Mice (C57BL/6 J obtained from JAXTM) were housed 2–5 per cage under a 12-hr light-dark cycle with ad libitum access to food and water. All animal experiments were approved by Thuringian state authorities and performed in accordance with European Commission regulations for the care and use of laboratory animals. Our study is reported in accordance with ARRIVE guidelines28. In all experiments, male and female mice aged 8–30 weeks between 25 and 35 g body weight were used.

Drugs and routes of administration

All drugs were freshly prepared prior to use and were injected subcutaneously (s.c.) or intraperitoneally (i.p.) in unanaesthetized mice at a volume of 10 μl/g bodyweight. Opioid drugs were diluted in 0.9% (w/v) saline and cannabinoid drugs in vehicle solution, containing 13.2% ethanol and 0.25% Tween80 in 0.9% saline, for injections. Drugs were obtained and used as follows: fentanyl citrate (0.3 mg/kg for 15 min; s.c.) (B. Braun 06900650), levomethadone hydrochloride (15 mg/kg for 30 min; s.c.) (Sanofi-Aventis 07480196), naloxone (2 mg/kg for 25 min; s.c.) (Ratiopharm 04788930), CP-55940 (0.75 mg/kg for 30 min; i.p.) (Sigma-Aldrich C1112) and AM251 (3 mg/kg for 45 min; i.p.) (MedChemExpress 183232-66-8). A total of 35 mice (n = 4–6 per treatment condition) were used.

Immunohistochemistry

Mice were deeply anaesthetized with isoflurane (CP-Pharma 4001404) and then subjected to a transcardial perfusion with calcium-free Tyrode’s solution containing protein phosphatase-inhibitors (+PPIs) (1 tablet PhosSTOP per 10 ml) (Roche 04906845001) followed by Zamboni’s fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 7.4 +PPIs. Brains and spinal cords were rapidly dissected and postfixed in the same fixative for 4 h at room temperature. The tissue was cryoprotected by immersion in 10% sucrose +PPIs followed by 30% sucrose +PPIs for 48 h at 4 °C before sectioning using a freezing microtome. Tissue was cut into 40 μm sections. Free-floating sections were washed in PBS with or without (+/-) PPIs (depending on the experiment and investigated GPCR) and incubated in methanol containing 0.3% H2O2 for 30 min. After washing in PBS-T (PBS + 0,3% Tween®20), the sections were blocked in PBS containing 0.3% Triton X-100, +/- PPIs and 10% NGS for 2 h. Subsequently, the sections were incubated with primary antibody in PBS containing 0.3% Triton X-100, 2% NGS + /- PPI overnight at 4 °C. Where indicated, primary antibodies were preincubated with 1 µg/ml of their cognate phospho-peptide or the corresponding non-phospho-peptide for 1 h at room temperature. When necessary, staining of primary antibody was detected using the biotin amplification procedure as described29, 30. Briefly, tissue sections were transferred to biotinylated donkey anti-rabbit IgG (1:300 in PBS containing 0.3% Triton X-100, +/-PPIs and 10% NGS) for 2 h, washed in PBS-T + /-s PPIs and then incubated in AB solution (reagents from the Vector ABC kit; 25 μl A and 25 μl B in 10 ml PBS + 0.3% Triton X-100 + /- PPIs) for 60 min, washed again in PBS-T + /- PPIs and transferred to biotinylated tyramine (BT) solution (BT was prepared as described by Adams31, 5 μl BT + 0.01% H2O2 in 1 ml PBS + 0.3% Triton X-100 and + /- PPIs) for 20 min, followed by a final incubation step in streptavidin-AlexaFluor555 conjugate 1:400 in PBS + 0.3% Triton X-100 containing 10% NDS +/− PPI) overnight at 4 °C. For double-staining procedures, the primary np-AB was incubated overnight as indicated and after several washing steps on the following day conjugated to the secondary AB, again overnight. Sections were then mounted onto SuperFrost Plus glass slides (ThermoFisher 15438060) and cover slipped with Eukitt (ORSAtec). Specimens were examined using a Zeiss LSM 900 laser scanning confocal microscope equipped with ZEN software for image analysis.

Western Blot and immunoprecipitation

Wildtyp mice were treated with agonist or vehicle. Mice were anesthetized with isoflurane, killed by cervical dislocation, and brains were quickly dissected, excluding the cerebellum. Brain samples were immediately frozen in liquid nitrogen. Brains were transferred to ice-cold detergent buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), containing protease and phosphatase inhibitors), homogenized, and centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant was then immunoprecipitated with the polyclonal goat np-CB1 antibody bound to protein A-agarose beads (20333, ThermoScientific, Germany) for 90 min at 4 °C. Proteins were eluted from the beads with SDS sample buffer for 25 min at 43 °C and then resolved on 8% SDS-polyacrylamide gels. After electroblotting, membranes were incubated with pS425-MOP antibody, followed by detection using a chemiluminescence detection system. Blots were subsequently stripped and incubated again with the rabbit np-CB1 antibody to confirm equal loading of the gels.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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