Cresyl Violet Staining of Tissue Sections: A Staining Procedure to Visualize Cartilage and Bones in Frozen Mouse Embryo Sections

Protocolo

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

1. Prepare solutions for staining and dehydration.
   1. 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.
   2. Place 45 mL of 95% ethanol in a 50 mL centrifuge tube on ice.
   3. Place 45 mL of 100% ethanol in a 50 mL centrifuge tube on ice.
   4. Place 45 mL of xylene in a 50 mL centrifuge tube at room temperature.
      CAUTION: Xylene should be used in a fume hood.
2. 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.
3. 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.
4. Stand slides on a delicate task wiper for 5 min to drain xylene and dry sections.

Materiais

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