The vesicles enclosed by a single membrane in animal cells belong to the vacuolar system, such as lysosomes, Golgi complexes, endoplasmic reticula, phagosomes, transport vesicles and so on. The vacuolar system is particularly well-developed in chondrocytes. A similar vacuolar system also exists in plant cells, which is generally acidic under normal conditions. Neutral Red is a weak basic dye in the form of red powder and also a pH indicator with a color change range of pH 6.4–8.0 (turning from red to yellow). In a neutral or slightly alkaline environment, living plant cells can absorb a large amount of Neutral Red and secrete it into the vacuolar system. Since the nucleic acids in the nucleus and the vacuolar system are both acidic, the Neutral Red entering them dissociates a large number of cations and exhibits a cherry red color. Thus, the nucleus and vacuolar system are stained red, while the protoplasm and cell wall are generally unstained. In dead cells, due to the denaturation and coagulation of protoplasm, the cell sap cannot be retained in the vacuolar system, so the vacuolar system will not be stained after Neutral Red treatment. On the contrary, the cations of Neutral Red bind to the protoplasm and cell nucleus with a certain negative charge, resulting in the staining of protoplasm and cell nucleus. Methylene Blue is a weak acidic dye soluble in water and alcohol, and it is widely used in the preparation techniques of animal and plant specimens. Combined with Eosin, it can stain nerve cells, and it is also an indispensable dye in bacterial specimen preparation; its aqueous solution serves as a vital stain for protozoa. Methylene Blue is extremely susceptible to oxidation, so specimens stained with it cannot be preserved for a long time.
Neutral Red-Methyl Blue Stain Solution is a mixture of 1% Neutral Red and Methylene Blue, used to identify cell viability. This reagent stains the nucleus and vacuolar system of living cells red, the cytoplasm of dead cells blue, and the cell nucleus of dead cells dark blue, and is commonly used for the staining of plant samples.
Operating Procedures (For Reference Only):
(I) Plant Tissues
1. Take the plant root tip, place it on a glass slide, and make a longitudinal cut with a knife.
2. Add a drop of Neutral Red-Methylene Blue Staining Solution and stain for 3–8 minutes. Aspirate the staining solution and add one drop of Ringer’s solution or normal saline.
3. Cover with a coverslip and press it gently with forceps (to flatten the root tip for easy observation), then observe or photograph under a light microscope.
(II) Cell Samples
1. Wash the cell samples with PBS or normal saline, centrifuge at 2000 r/min for 10 minutes, and resuspend the pellet in PBS.
2. Add an equal volume of Neutral Red-Methylene Blue Staining Solution, mix well, and stain for 2–5 minutes.
3. Aspirate the mixture, drop it onto a glass slide, cover with a coverslip, and then observe or photograph.
Staining Results:
Live cells: The nucleus and vacuolar system appear red.
Dead cells: The cytoplasm appears blue, and the nucleus appears dark blue.
Precautions:
1. It is recommended to conduct a pre-experiment with 1–2 samples when using this reagent for the first time.
2. A small amount of precipitate may form in the Neutral Red-Methylene Blue Staining Solution after long-term storage, which can be filtered with a cell strainer (no impact on staining effect).
3. For the staining of cell samples, the cell pellet can be mixed with the staining solution before preparing a smear for observation.
4. The staining time can be adjusted according to the staining results and experimental requirements during sample staining.
5. The staining time of Neutral Red Staining Solution can be adjusted according to the staining results and experimental requirements.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
7. This product is for research use only and shall not be used for any other purposes.
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