Goat anti guinea pig igg

Immunohistochemical reactions for electron microscopy were carried out using the pre-embedding immunogold method described previously (Luján et al., 1996 (link)). Briefly, free-floating sections were incubated in 10% (v/v) NGS diluted in TBS. Sections were then incubated in anti-Cav3.1 or anti-Cav3.2 antibodies [3–5 μg/mL diluted in TBS containing 1% (v/v) NGS], followed by incubation in goat anti-guinea pig IgG or anti-mouse IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY, USA), respectively. Sections were postfixed in 1% (v/v) glutaraldehyde and washed in double-distilled water, followed by silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes Inc., Stony Brook, NY, USA). Sections were then treated with osmium tetraoxide (1% in 0.1 M PB), block-stained with uranyl acetate, dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan (Fluka) resin. Regions of interest were cut at 70–90 nm on an ultramicrotome (Reichert Ultracut E, Leica, Austria) and collected on the single slot pioloform-coated copper grids. Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds’s lead citrate. Ultrastructural analyses were performed in a Jeol-1010 electron microscope.