Immunohistochemistry

We use the immunoperoxidase approach primarily for its sensitivity and stability. The chromogen of choice is 3,3′-diaminobenzidine (DAB). This approach has been optimized to reveal the regional distribution of a given protein at the light microscopic level, but can also be exploited to expose the subcellular distribution at the electron microscopic level. For light microscopic analysis, specific regions of the CNS are analyzed and photographed using a Nikon Eclipse 80i upright microscope  and a Nikon DS-Fi1 digital color camera.

Immunofluorescence is regularly exploited in the lab for its expedient ability to reveal the cellular expression pattern of more than one protein in the same sample. Our Nikon Eclipse 80i upright microscope is equipped with an epi-fluorescent attachment, containing FITC, Texas Red, DAPI and Cy5 filter cubes, plus a Nikon Super High Pressure Mercury lamp. However, most of the detailed analysis is performed on the Nikon A1R confocal system, which is based on a Nikon Ti-E inverted microscope. It is equipped with 4 lasers providing 405, 458, 488, 514, 561, 641 nm laser lines as well as with a 32-channel spectral detector, greatly enhancing our flexibility in the design of multi-coloured staining experiments. Notably, recent technological advances also offer that multiple targets can be studied at the subcellular level. We are routinely using the STORM super-resolution technology to uncover the subcellular distribution of target proteins with a 20-40 nm resolution.

Finally, the immunogold labeling technology is used to reveal the precise subcellular position of a given protein at a nanometer scale. Target proteins in fixed brain sections are bound by antibodies conjugated to 0.8 nm gold particles, which are further enhanced using a silver intensification approach. The immunostained sections are then treated with OsO₄ and uranyl acetate, dehydrated in an ascending series of ethanol and acetonitrile, and embedded in Durcupan. Areas of interest are re-embedded and re-sectioned. Electron microscopic analysis of 20-60 nm thick ultra-thin sections on Formvar-coated single-slot grids is performed using a Hitachi 7100 electron microscope. Electron micrographs are taken at 1,000 – 100,000x magnification