CB2 receptors were immunostained with the polyclonal anti-cannabinoid receptor II antibody (ab45942, Abcam, USA; dilution 1:50), using Alexa 488-conjugated anti-rabbit raised in goat (Molecular Probes–Invitrogen; dilution 1:400) as the secondary antibody. Antibodies were diluted in PBS supplemented with 1% bovine serum albumin (BSA) and 0.04% triton. All preparations were contrasted with TOPRO-3 iodide (Life Technologies, USA; dilution 1:500). Specificity was confirmed in separated experiments with additional negative controls, including tissue sections incubated with primary antibody pre-adsorbed to an excess of control peptide (ab45941, Abcam, USA; dilution 1:25).
Cochleae assigned to surface preparations were immunolabeled once bisected into two halves and before final dissection, by means of a “free floating” technique. Transversal sections were immunolabeled once mounted on the slide and conveniently dried.
As a positive control of the primary antibody used, spleen and kidneys [28 (link)] of each animal were submitted to the same protocol.
Cochleae assigned to surface preparations were immunolabeled once bisected into two halves and before final dissection, by means of a “free floating” technique. Transversal sections were immunolabeled once mounted on the slide and conveniently dried.
As a positive control of the primary antibody used, spleen and kidneys [28 (link)] of each animal were submitted to the same protocol.
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