Nanogold anti rabbit igg

Samples were fixed in 1% PFA/2.5% GA in 0.1PB containing 3% sucrose and 1 mM MgSO₄, followed by dehydration in graded methanol and acetone, embedded in Eponate and cut to a 90 nm thickness on a Leica Ultracut microtome. Serial sections were probed using antibodies targeting small molecules L-aspartate, L-glutamate, glycine, L-glutamine, γ-aminobutyric acid (GABA). Antibodies incubated overnight at room temperature were visualized with goat anti-rabbit secondary IgG coated with 1.4 nm gold (Nanoprobes Nanogold® -anti Rabbit IgG) and silver intensified for CMP^{41 (link)}. 8-bit images (243 nm/pixel) were mosaicked and registered with ir-tweak (https://www.sci.utah.edu/download/ncrtoolset.html)^{40 (link)}. RGB channels for display images were linearly contrast stretched for display. Molecular signals were visualized as rgb maps (e.g., γGE → rgb assigns γ-aminobutyric acid, glycine and L-glutamate to red, green, and blue color channels, respectively). For display only, raw data channels were linearly contrast-stretched and sharpened with unsharp masking. Monochrome and RGC images were intensity mapped.

Mouse muscle tissues were dissected and immediately fixed with 0.2% glutaraldehyde, 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, at 4 °C. The tissues were then sectioned into 150-µm-thick sections using a Leica Vibratome. Pre-embedding immune-labelling was performed on the Vibratome sections using rabbit anti-STX4 primary antibody (Millipore Sigma #AB5330) and Nanogold® anti-rabbit IgG (Nanoprobes) diluted 1:100 in PBS for 1.5 h. HQ Silver enhancement kit (Nanoprobes) was used to further develop the Nanogold® staining. The Vibratome sections were then processed following standard sample preparation for TEM and examined as described above.