Eosin Staining of Soft-Tissue Samples: An Eosin-Based Procedure for Whole Mouse Kidney Staining to Visualize Specific Tissue Microstructures
Transcript
For cell cytoplasm-specific staining in soft tissue microenvironments, obtain a freshly harvested mouse kidney. Treat it with a fixative solution containing formaldehyde, glacial acetic acid, and methanol. Incubate at low temperatures.
Formaldehyde penetrates the cells, crosslinking proteins to proteins and proteins to nucleic acids. Methanol displaces water from the tissue and coagulates proteins. Acetic acid causes intracellular acidification and tissue swelling, thereby counteracting tissue shrinkage caused by methanol.
Fixation inactivates enzymes and stabilizes tissue morphology for subsequent staining. Remove excess fixatives using buffer. Incubate the tissue with eosin Y, an acidic dye.
Eosin penetrates fixed cells and enters the cytoplasm. The acidic environment of the cytoplasm, from acetic acid treatment, causes maximum protonation of amino groups of cytoplasmic proteins.
Eosin, with negatively-charged functional groups, electrostatically interacts with protonated groups of proteins, forming a stable dye-protein complex, and stains the cytoplasm pink upon accumulation. Post-incubation, cut the stained kidney into small pieces, followed by tissue dehydration in increasing ethanol concentrations.
Embed the kidney tissue in paraffin wax. Obtain thin tissue sections using a microtome. Further, rehydrate the sections for microscopic analysis.
Unstained cell nuclei appear white against a pink-stained cytoplasm, enabling visualization of kidney microstructures such as proximal convoluted tubules, comprising epithelial cells with brush borders, or collecting ducts, which are lined with flattened epithelial cells without brush borders.
