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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Fixation and Staining of Cryopreserved Brain Tissue Sections
- To preserve RNA integrity, clean all instruments to be used with RNase cleaning solution. Proceed with the fixation and staining protocol inside a fume hood.
- Prepare the described fixative solutions in clean RNase-free 50 mL tubes. Make all solutions with RNase-free water on the day of the staining.
- Same day the laser microdissection will be performed, prepare 100%, 95%, 70%, and 50% ethanol solutions. Keep the solutions in tightly closed tubes at room temperature.
- Prepare 4% Cresyl violet and 0.5% eosin Y in 75% ethanol solution. Vortex the solution vigorously for 1 min and filter them through a 0.45 µm nylon filter to eliminate traces of undissolved powder.
- Place the tissue slides into a container with 95% ethanol for 30 s. Transfer slides to the tube containing 75% ethanol; leave slides there for 30 s.
- Transfer slides to 50% ethanol and leave them there for 25 s. At this point, the OCT will be dissolved. Transfer the slide to 4% Cresyl violet solution for 20 s and then transfer to 0.5% eosin Y solution for 5 s.
- Take the slide out of the dye solution and blot the slide dry with a filter paper. Then, place the slides in 50% ethanol for 25 s. Transfer the slides to 75% ethanol for 25 s. Transfer the slides to 95% ethanol for 30 s. Transfer the slides to 100% ethanol for 60 s.
- Rinse the slide with xylene. Transfer them to a container with xylene and wait 3 min.
- Prepare mounting medium (e.g., Pinpoint gum) in RNase-free water. To mount mouse brain sections, dilute the mounting medium in RNase-free water at a ratio of 1:10.
- Dry the slides on an RNase-free surface at room temperature for 10 s. Before the xylene dries, proceed to mounting the slides with the tissue sections.
- Gently disperse mounting solution on top of the tissue on the slide with a sterile and RNase-free thin paintbrush. Wait 10–20 s and then immediately transfer the tissue slides to the microscope microdissection platform.
NOTE: The ratio of mounting medium to RNase-free water used for mounting varies depending on the tissue of interest. The mounting medium/water ratio preserves glioma tissue morphology for laser microdissection without affecting the RNA integrity.