This article details a protocol for differentiating specific cell populations in heterogeneous mouse brain tumor tissue for isolation. The method involves sequential staining and dehydration steps to prepare the tissue 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.
Submerge the slides in 30-second sequential descending ethanol immersions, followed by crystal violet solution staining for 20 seconds and 5 seconds in 0.5% eosin Y solution.
At the end of the eosin Y incubation, use filter paper to blot the slides dry and dehydrate the slides in sequential ascending ethanol immersions. After the 60-second ethanol immersion, rinse the slides in a container of xylene for 3 minutes.
Next, dry the slides on RNase-free surface at room temperature for 10 seconds before mounting the samples in mounting medium prepared with RNase-free water. After 10 to 20 seconds, transfer the slides onto the microscope microdissection platform.
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