Neutral Red is a weakly basic pH indicator with a transition range of pH 6.4–8.0 (from red to yellow). In neutral or slightly alkaline environments, living plant cells can absorb a large amount of Neutral Red and secrete it into the vacuoles. Since vacuoles are generally acidic, Neutral Red entering the vacuoles dissociates into numerous cations and exhibits a cherry-red color; under these conditions, the protoplasm and cell walls are generally unstained. In dead cells, the protoplasm denatures and coagulates, and cell fluid cannot be retained in the vacuoles. After staining with Neutral Red, no vacuolar coloration occurs. Instead, the cations of Neutral Red bind to the negatively charged protoplasm and nuclei, resulting in staining of the protoplasm and nuclei.
Neutral Red Ethanolic Staining Solution (0.1%) is a commonly used reagent for tissue or cell staining that stains nuclei red, and is frequently used for mast cell staining.
Protocol
I. Sample Preparation
1. For Paraffin Sections
(1) Deparaffinize in xylene for 5–10 min.
(2) Replace with fresh xylene and deparaffinize for another 5–10 min.
(3) Absolute ethanol for 5 min.
(4) 90% ethanol for 2 min.
(5) 70% ethanol for 2 min.
(6) Distilled water for 2 min.
2. For Frozen Sections
Equilibrate to room temperature, then rinse with distilled water for 2 min.
3. For Cultured Cells
(1) Fix with 4% paraformaldehyde for ≥10 min.
(2) Rinse with distilled water for 2 min.
(3) Replace with fresh distilled water and rinse for another 2 min.
II. Neutral Red Staining
1. Stain the prepared samples with Neutral Red Ethanolic Staining Solution (0.1%) for 0.5–1 h.
2. Wash thoroughly with distilled or tap water.
3. Observe and photograph.
Staining Result
Precautions
If dehydration, clearing and mounting are required, prepare xylene, neutral balsam or other mounting media by yourself. For paraffin sections, prepare 70% and 90% ethanol, absolute ethanol and xylene by yourself.
It is recommended to perform a pilot experiment on 1–2 samples when using this reagent for the first time.
The staining time with Neutral Red Ethanolic Staining Solution (0.1%) can be adjusted according to the staining results and requirements.
For your safety and health, please wear a lab coat and disposable gloves during operation.
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