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

To differentiate specific cell populations in a heterogeneous mouse brain tumor tissue for their subsequent isolation, begin by taking a glass slide carrying the cryopreserved brain tumor tissue section of interest.

Sequentially incubate the tissue in decreasing concentrations of ethanol. Ethanol dissolves the embedding medium that has penetrated the tissue and fixes the tissue while maintaining nucleic acid integrity. 

Additionally, ethanol rehydrates the tissue, enabling even staining during the subsequent steps.

Now, treat the tissue with Cresyl violet. Cresyl violet - a basic dye - stains acidic cellular components such as nucleic acids.

Thereafter, counterstain the tissue with Eosin Y. Eosin Y - an acidic dye - stains basic cellular components such as cytoplasmic proteins.

This differential staining step helps distinguish between the cytoplasm and other cellular structures, enabling the identification of cells of interest.

Post staining, immerse the tissue in increasing concentrations of ethanol to dehydrate the tissue and avoid excessive tissue distortion.

Next, wash the tissue with xylene. Xylene displaces and removes ethanol from the tissue section.

Finally, add a suitable mounting solution onto the tissue section to preserve tissue morphology.

The processed tissue section is ready to be used for laser microdissection - a process that allows for precise isolation of specific cells from the tissue.