Z-stack through the CA region of the hippocampus of adult rat, immunostained to reveal dendrites of neurons (red), astroglia (green) and all nuclei (blue). This kind of approach can reveal the spatial relationships between the nonneuronal glial cells and neurons.
Tissue was perfused with 4% paraformaldehyde in PBS, postfixed overnight, sectioned at a thickness of 25 µm,incubated in blocking solution (0/25% lambda carageenen, 1%bovine serum albumin, and 0.5% Triton X-100 in 0.01M PBS) and immunostained using antibodies to the dendritically-localized microtubule protein MAP2 (polyclonal Ab266 from S. Halpain), and the astroglial cytoskeletal protein GFAP (glial fibrillary acidic protein, monoclonal clone G-A-5, Sigma) followed by fluorescently-conjugated secondary antibodies (anti-mouse Alexa 488, 1:400; anti-rabbit Alexa 546, both from Molecular Probes), and incubated in 1:1000 Hoescht33258 to stain nuclei. A multiframe data set of 5x3x20(X,Y,Z) was acquired using the Leica SP5 confocal microscope with line-by-line sequential scanning, a 63X HCX PL APOCHROMAT, 1.32NA, with 1.7 zoom at z-intervals of 0.5 µm, and saved as a tif image stack.
|Spatial Axis||Image Size||Pixel Size|