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Immunohistochemistry
Tissue Preparation
Brown Norway rats were euthanized and perfused with 0.1M phosphate buffer saline (PBS), followed by 4% paraformaldehyde (PFA) in 0.1M PBS. The eyes were enucleated and post-fixed for 24 hours at 4°C. After fixation the eyes were cryoprotected in 30% sucrose, embedded in gelatine, post fixed again in PFA and sectioned at 30 μm on a freezing microtone.
Immunohistochemistry
Eye sections were washed with phosphate buffer saline (PBS) and permeabilized with 3% Triton x-100. Following the wash, the tissue was exposed to 10% donkey serum for 1 hour, in order to block non-specific binding. Then, the tissue was incubated with the primary antibody to the CB1 receptor (Chemicon International, Temecula, CA, USA) at 4°C overnight. The antibody stock (2μg/ml) was diluted to 1:300. The labelling of CB1 receptors was visualized with the aim of a fluorescent secondary antibody (donkey anti-rabbit IgG secondary conjugated to Alexa 488; Molecular Probes, Eugene, OR, USA). Finally, the sections were mounted in an aqueous mount and the CB1 receptor labelling was viewed using a confocal microscope.
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
RT-PCR technique was used to determine the mRNA expression of the CB1 receptors in rat ciliary body epithelium. The primers for amplification of the human CB1 receptors were: 5’- TGCAGGCCTTCTTACCACTTCATC-3’ (forward, bp 536-559) and 5’-GACGTGTGGATGATGATGCTCTTC-3’ (reverse, bp 1056-1033) as stated in GenBank#:XM 004350. PCR conditions for amplification of the CB1 receptor included: denaturation of the primers at 94°C for 1 minute, 35 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute and extension at 72°C for 1minute, followed by final extension at 72°C for 7 minutes. RT-PCR amplification of cyclophilin was also performed as the internal control and used to normalize the CB1 receptor for the mRNA expression. PCR reactions were carried out using a PCRExpress Thermal cycler (Hybaid, Franklin, MA, USA). RT-PCR amplification, with a primers pair specific for human CB1 receptors (cDNA), detected a PCR product with a predicted size of 520 bps. The PCR product obtained for the hCB1 receptor was cloned and sequenced. The oligonucleotide sequence was identical to that of the hCB1 receptor, cDNA, reported in GenBank (GenBank#: XM 004350).
Western Blot Analysis
The ciliary body epithelium obtained from rat eyes was disrupted and homogenized in a lysis buffer and protease inhibitor with a dounce homogenizer, maintaining a temperature of 4°C throughout the procedure. Protein analysis was performed using Micro BCATM Protein Assay Reagent Kit (Chromotographic Specialities, Brockville, Ontario). 25-50 μg of the samples were separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) in a running buffer for approximately 1 hour at 100V and transferred to an ImmobilonTM-P PVDF membrane (Millipore Corporation, Bedford, MA, USA) overnight at 35 V. The membrane was then air dried at room temperature for 2 hours. It was then incubated with the polyclonal rabbit anti-human cannabinoid CB1 receptor antibody (1:300 dilution) overnight in a shaker at 4°C. Three washes with a PBS-Tween 80 (containing a 2% Tween 80) for 20 minutes followed. The membrane was incubated with anti-rabbit peroxidase with conjugated IgG for 1 hour at 37°C on a rotator. Again, the membrane was washed with PBS-Tween 80 buffer for 20 minutes, three times. The immunoreactive proteins were visualized by chemiluminescence using an ECL plus system (Amersham Life Science, Little Chalfont, Buckinghamshire, England).