Visualization of a Mouse Brain Choroid Plexus Using Scanning Electron Microscopy

Samenvatting

Source: Van Wonterghem, E., et al. Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus. J. Vis. Exp. (2022)

This video demonstrates the preparation and imaging of choroid plexus tissue from a mouse brain using scanning electron microscopy (SEM). The process includes tissue fixation, dehydration, and coating with a conductive platinum layer to stabilize and prepare the sample for imaging. SEM captures detailed images of epithelial cells with microvilli, providing insights into the tissue’s structural features.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Morphological analysis of choroid plexus (CP) tissue using scanning electron microscopy (SEM)

CAUTION: Toxic solutions are used in the following processing steps. It is recommended to perform the sample preparation in a fume hood.

1. Perform sample preparation for SEM as described below.
   1. Transfer the fresh isolated CP into freshly made fixation solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4). Incubate overnight at 4 °C.
      NOTE: As the CP tissue is very fragile and thin, the tissue should be put in small specimen baskets (see Table of Materials) to transfer it between buffers. In this way, the structure of the tissue will be better preserved. A cacodylate-based fixative is used over a phosphate-buffered saline (PBS)-based one, as the strong cacodylate buffer can counter the low pH of glutaraldehyde in the EM fixative.
   2. Wash the sample 3x for 5 min each with 3-5 mL of 0.1 M Na-cacodylate buffer (pH 7.4).
   3. Post-fix the samples in 3-5 mL of 2% osmium tetroxide in 0.1 M Na-cacodylate buffer for 30 min. Wash the samples 3x for 5 min each with 3-5 mL of ultrapure water.
   4. Dehydrate the samples in a series of ice-cold solutions of increasing ethanol (EtOH) concentrations (50%, 70%, 85%, 100%), for 15 min per EtOH solution. Use a critical point dryer to properly point dry the sample.NOTE: Changing from liquid to gas phase affects the surface tension, causing damage to the surface structure. The critical point drying step allows for the preservation of the surface structure.
   5. Position the sample carefully on a specimen mount provided with a carbon sticker (see Table of Materials).
   6. Coat the samples with a thin layer (2-5 nm) of platinum. To do so, mount a platinum source in a vacuum system between two high-current electrical terminals and heat the platinum to its evaporation temperature. A fine stream of platinum is deposited on the sample.NOTE: Besides platinum, gold or gold/palladium can also be used.
2. Visualize the CP samples with SEM (see Table of Materials). The visualization procedure of CP tissue via SEM is similar to that of other types of tissues and depends on the software used and instruments.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Aluminium specimen mounts	EM Sciences	75220
Cacodylate buffer	EM Sciences	11652
Carbon adhesive tabs -12 mm	EM Sciences	77825-12
Critical point dryer	Bal-Tec	CPD030
Crossbeam 540	Zeiss		SEM system
Forceps	Fine Science Tools GmbH	91197-00
Glutaraldehyde	EM Sciences	16220
Osmium Tetroxide	EM Sciences	19170
Paraformaldehyde	Sigma-Aldrich	P6148
Phosphate buffered saline (PBS)	Lonza	BE17-516F
Platinum	Quorum	Q150T ES	PBS without Ca⁺⁺, Mg⁺⁺ or phenol red; sterile filtered
Specimen Basket Stainless Steel	EM Sciences	70190-01