5.2. Preembedding immunogold method for electron microscopy

Tissue sections were pre‐incubated in a blocking solution containing 10% bovine serum albumin (BSA; Sigma‐Aldrich, St. Louis, MO), 0.1% sodium azide and 0.02% saponin in Tris–HCl buffered saline (TBS; pH 7.4) for 30 min at room temperature (RT). Subsequently, sections were incubated with a polyclonal rabbit anti‐CB₁ receptor antibody (1:500; ImmunoGenes, Budapest, Hungary) prepared in blocking solution with 0.004% saponin for 2 days at 4°C. After several washes in 1% BSA/TBS, tissue sections were incubated in a secondary 1.4 nm gold‐labeled goat anti‐rabbit IgG (1:200; Nanoprobes Inc., Yaphank, NY) prepared in the washing solution with 0.1% sodium azide and 0.004% saponin for 4 h at RT. Tissue was washed overnight in 1% BSA/TBS at 4°C, postfixed in 1% glutaraldehyde in TBS for 10 min at RT and washed in double‐distilled water (ddH₂0). Gold particles were silver‐intensified with a HQ Silver kit (Nanoprobes) in the dark for 12 min and tissue was washed with ddH20 followed by 0.1 M PB. The day after, sections were osmicated (1% OsO₄ in 0.1 M PB; pH 7.4) for 30 min. After 3 × 10 min washes in 0.1 M PB, tissue sections were dehydrated in graded ethanol concentrations (50%–100%) to propylene oxide and embedded in epoxy resin (Sigma‐Aldrich) by immersion in decreasing concentration of propylene oxide (1:3 for 30 min, 1:1 for 1 h and 3:1 for 2 h). Tissue was then embedded in fresh resin overnight and allowed to polymerize at 60°C for 2 days. Following visualization at the light microscope, selected tissue portions were trimmed and glued onto epoxy resin capsules. Semi‐thin sections (500 nm‐thick) were cut from epoxy blocks using a Power Tome ultramicrotome (RMC Boeckeler, Tucson, AZ) and stained with 1% toluidine blue. Ultrathin (50–60 nm‐thick) sections were then cut with a diamond knife (Diatome, Hatfield PA), collected on nickel mesh grids and stained with 4% uranyl acetate for 30 min and 2.5% lead citrate for electron microscope visualization.