Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Overview

This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection.

Protocol

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

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Rinse the slide with xylene. Transfer them to a container with xylene and wait 3 min.
9. 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.
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.
11. 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.

Materials

Name	Company	Catalog Number	Comments
Cresyl Violet Acetate	Sigma Aldrich	C5042
Eosin Y	Sigma Aldrich	E4009
RNaseZap RNase Decontamination Solution	Fisher Scientific	AM9780
PEN Membrane Glass Slide (2 µm)	Lieca	1150518
Pinpoint Solution	Zymo Research	D3001-1
Tissue-Plus O.C.T. Compound	Fisher Scientific	23-730-571