Rabbit anti cb1r

Neurons were fixed in 4% PFA, then coverslips were treated for 10 min with 50 mM NH4Cl and incubated in blocking buffer (0.22% gelatin, 0.1% Triton X-100 in PBS) for 30 min, before incubation with primary antibodies for 1 h at room temperature in blocking buffer. For CB1R staining, neurons were incubated for 30 min at 37°C with anti-CB1R antibody, then rinsed three times and fixed to continue the procedure with other antibody staining. The primary antibodies used were: chicken anti-MAP2 (1:10,000, Abcam), mouse anti-ankyrinG (1:100) from NeuroMab, anti-Tau-1 from Millipore (1:1000) and rabbit anti-CB1R (1:50) from Cayman (Cat. 101500). The secondary antibodies used were a donkey anti-mouse, anti-rabbit or anti-chicken Alexa-Fluor 488, 594, or 647 (1:1000). Phalloidin Alexa-Fluor 594 was used at a concentration of 1:100. Nuclei were stained using 4′,6-diamidino-2-phenylindole, and coverslips were mounted in Fluoromount G. Images were acquired on a vertical Axioskop-2 plus microscope (Zeiss) or a Leica SP5 confocal microscope under the same conditions to compare intensities. Figures were prepared for presentation using the Adobe CS4 software.

Tissue sections and astrocyte cultures obtained from rat, wild type and CB₁-R knock out mice spinal cords were first incubated with a mixture of antibodies that contained mouse anti-GFAP (1:1000, catalog no.: MAB3402, Millipore, Temecula, California, USA), rabbit anti-CB₁-R (1:1000, catalog no.: 10006590, Cayman Chemical, Ann Arbor, Michigan, USA) and goat anti-DGLα (1:500, catalog no.: Af1080, Frontier Institute, Hokkaido, Japan). The sections and cultures were then transferred for overnight incubation in a mixture of donkey anti-mouse IgG conjugated with Alexa Fluor 488 (1:1000, catalog no.: A-21202, Invitrogen, Eugene, Oregon, USA), donkey anti-rabbit IgG conjugated with Alexa Fluor 647 (1:1000, catalog no.: A-31573, Invitrogen, Eugene, Oregon, USA) and donkey anti-goat IgG conjugated with Alexa Fluor 555 (1:1000, catalog no.: A-21432, Invitrogen, Eugene, Oregon, USA) secondary antibodies. Before the antibody incubations, the sections were kept in 10% normal donkey serum (catalog no.: ab7475, Abcam, Cambridge, UK) for 50 minutes. Antibodies were diluted in 10 mM TPBS (pH 7.4) containing 1% normal donkey serum (catalog no.: ab7475, Abcam, Cambridge, UK). Sections were mounted on glass slides and covered with VectaShield-DAPI (catalog no.: H-1200, Vector Laboratories., Burlingame, California, USA).