This article details a method for cell cytoplasm-specific staining in soft tissue microenvironments, specifically using mouse kidney samples. The process involves fixation, staining with eosin Y, and preparing tissue sections for microscopic analysis.
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.
Start by fixating the soft tissue samples. Fill a 50-milliliter centrifuge tube with a fixative solution containing 9.5 milliliters of 4% formaldehyde and 0.5 milliliters of glacial acetic acid. Add the soft tissue sample to the centrifuge tube, and refrigerate it for 24 to 72 hours.
After refrigeration, wash the soft tissue sample with DPBS solution for 1 hour. Then, stain the sample by placing it in 2 milliliters of eosin Y-staining solution, and incubate it on a horizontal shaking plate for 24 hours.
On the next day, carefully take the soft tissue sample out of the sample container and remove excess staining agent with cellulose tissue paper. Place it in a conical container above an ethanol vapor phase for storage and further use.
繁體中文
