This article details the preparation of choroid plexus tissue from mouse brains for scanning electron microscopy (SEM). The process involves fixation, dehydration, and coating to ensure high-quality imaging of epithelial cells with microvilli.
Take the choroid plexus from a mouse brain in a specimen basket.
The tissue, located within the brain ventricles, contains a layer of epithelial cells featuring microvilli on their apical surface.
Incubate the tissue in a fixative solution to preserve its cellular architecture.
Wash with a buffer to remove excess fixative and stabilize the pH.
Incubate the tissue with osmium tetroxide that binds to cell membrane lipids, preserving membrane stability, then rinse off excess reagent.
Dehydrate the tissue using increasing alcohol concentrations to prevent tissue distortion during imaging.
Dry the tissue, secure it onto a carbon-coated specimen mount, then coat it with a conductive platinum layer.
Using scanning electron microscopy, focus an electron beam onto the surface. The platinum layer dissipates electrons, preventing charge buildup and minimizing imaging artifacts.
A detector captures the electrons backscattered from the surface, producing an image of the tissue exhibiting epithelial cells with microvilli.
To perform the sample preparation for SEM, transfer the freshly isolated choroid plexus into freshly made fixation solution, and incubate overnight at 4 degrees Celsius. Once done, wash the sample three times using 3 to 5 milliliters of 0.1 molar sodium cacodylate buffer for five minutes each.
Post-fix the samples in 3 to 5 milliliters of 2% osmium tetroxide in 0.1 molar sodium cacodylate buffer for 30 minutes. Then wash the samples three times for five minutes each using 3 to 5 milliliters of ultrapure water. Next, dehydrate the samples in an increasing series of ice-cold ethyl alcohol concentrations with 15 minutes per ethyl alcohol solution. Use a critical point dryer to dry the sample properly. Position the sample carefully on a specimen mount with a carbon sticker, and visualize the choroid plexus samples using SEM.
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