Overview
This video describes cresyl violet staining procedure to visualize bones and cartilage in coronal sections of frozen mouse embryos. This staining technique helps to differentiate bones and cartilage with distinct colors from the surrounding tissues.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sample Preparation for Laser Capture Microdissection
- Prepare solutions for staining and dehydration.
- Place 45 mL of 80% ethanol in each of three 50 mL centrifuge tubes on ice. Label them as #1, #2, and #3. Dilute ethanol with RNase/DNase free water.
- Place 45 mL of 95% ethanol in a 50 mL centrifuge tube on ice.
- Place 45 mL of 100% ethanol in a 50 mL centrifuge tube on ice.
- Place 45 mL of xylene in a 50 mL centrifuge tube at room temperature.
CAUTION: Xylene should be used in a fume hood.
- Thaw a PEN membrane slide briefly by placing it against a gloved hand, then wash twice for 30 s each in 45 mL of 80% ethanol (#1 and #2) with agitation in 50 mL centrifuge tubes to remove the OCT.
NOTE: It is important to remove OCT before staining, to prevent OCT loosened during staining from obscuring the sections. Forceps can be used to facilitate removal of OCT that is not washed off by agitation. - Lay slide on a sheet of aluminum foil. Pipette 0.8 mL of 0.1% cresyl violet in 50% ethanol onto the slide and stain for 30 s. Wash the slide in 45 mL of 80% ethanol (#3) for 30 s, and then dehydrate by passage for 30 s each through 45 mL of 95% ethanol, 100% ethanol, and xylene in 50 mL centrifuge tubes.
- Stand slides on a delicate task wiper for 5 min to drain xylene and dry sections.
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Materials
Name | Company | Catalog Number | Comments |
Centrifuge tube | ThermoFisher Scientific | 339653 | Conical sterile polypropylene centrifuge tubes, 50 mL |
Cresyl violet acetate | Sigma-Aldrich | C5042 | |
Delicate task wiper | ThermoFisher Scientific | 06-666 | |
Distilled water | Invitrogen | 10977-015 | DNase/RNase-Free |
Ethanol, absolute (200 proof) | ThermoFisher Scientific | BP2818 | Molecular biology grade |
Glass PEN membrane slide | Leica Microsystems | 11505158 | |
OCT compound | Electron Microscopy Sciences | 102094-106 | |
RNase decontamination agent | Sigma-Aldrich | R2020 | RNase decontamination agent for cleaning surfaces |
Xylene | Sigma-Aldrich | 214736 |