Permeabilization reagent, also known as permeabilization solution, typically achieves cell membrane permeabilization by using organic solvents (e.g., methanol, acetone) or detergents (e.g., Triton X-100, Saponin).
Organic solvents such as methanol or acetone can, on one hand, dissolve cell membranes and nuclear membranes to fully expose target proteins in the cytoplasm and nucleus; on the other hand, they can denature intracellular proteins to exert a fixation effect. Their advantage lies in simple operation—one-step treatment can achieve both fixation and permeabilization simultaneously. However, their disadvantages include the dissolution of membrane proteins and the hindrance to subsequent detection caused by the denaturation of some proteins. Therefore, organic solvents are relatively less used and only applicable to assays with rough requirements. Triton X-100 is a commonly used permeabilization reagent that can permeabilize cell membranes and nuclear membranes through the mechanism of non-specifically dissolving cell membranes. A drawback is that it is not conducive to membrane protein detection. Nevertheless, after cross-linking and fixation with paraformaldehyde, a considerable portion of membrane proteins will be cross-linked and immobilized, thus avoiding dissolution by Triton X-100 and enabling subsequent detection. Saponin can specifically dissolve cholesterol in cell membranes, thereby selectively creating pores on the cell membrane. Its advantages are suitability for membrane protein detection, especially for the detection of cell membrane marker proteins via flow cytometry. The disadvantages are poor permeabilization effect on cells with low cholesterol content, weaker permeabilization capability compared with Triton X-100 and organic solvents, and inability to permeabilize nuclear membranes and mitochondrial membranes with extremely low cholesterol levels. For lectin detection, permeabilization solutions containing non-specific detergents such as Triton X-100 exhibit significantly better performance than those mainly composed of Saponin.
PBS-Saponin Solution (Permeabilization Reagent, 0.5%), also referred to as immunostaining permeabilization solution, is mainly composed of 0.01M PBS (phosphate-buffered saline, pH 7.4) and 0.5% Saponin. It can be used for the permeabilization treatment of cell samples, frozen or paraffin sections in various in situ detection methods such as immunostaining. It helps expose action targets (e.g., antigens, nucleic acids) and facilitates the entry of antibodies, probes or labels into cells, thereby ensuring the detection effectiveness of staining and other assays. This product has strong permeabilization ability and is recommended for cell permeabilization in various routine assays including immunofluorescence, immunohistochemistry, immunocytochemistry and flow cytometry. It is particularly suitable for membrane protein detection. This product does not dissolve cell membranes and will not interfere with light scattering during flow cytometry analysis. However, it is not recommended for the detection of proteins in the nucleus and mitochondria. This product is a ready-to-use working solution and can be applied directly without dilution. The reagent is for research use only and not intended for clinical diagnosis or other purposes.
Operating Procedures (for reference only)
1. For sections: After completion of fixation and washing, add 50-100 μl of PBS-Saponin Solution dropwise to each sample. Alternatively, the sections can be completely immersed in a staining jar for permeabilization.
2. For cell samples: After completion of fixation and washing, add 1 ml of PBS-Saponin Solution to each well of a 6-well plate. For other multi-well plates, add the solution at a proportional volume.
3. For other samples: Add an amount of PBS-Saponin Solution sufficient to completely cover the samples.
4. Generally, incubate the samples in PBS-Saponin Solution at room temperature for 5-10 minutes to complete permeabilization. For samples that are difficult to permeabilize or when thorough permeabilization is required, the incubation time can be extended to 10-30 minutes at room temperature.
Precautions
1. For applications with low requirements for permeabilization effect, PBS-Saponin Solution (Permeabilization Reagent, 0.3%), PBS-Triton Solution (Permeabilization Reagent, 0.05%) or immunostaining washing solution can also be used.
2. This product is recommended for the detection of cell membrane proteins.
3. For applications requiring high permeabilization efficiency, PBS-Triton Solution (Permeabilization Reagent, 0.3%) or PBS-Triton Solution (Permeabilization Reagent, 0.5%) can be selected.
4. For lectin detection, PBS-Triton Solution (Permeabilization Reagent, 0.1%) or PBS-Triton Solution (Permeabilization Reagent, 0.3%) is recommended.
5. Precipitation may occur upon prolonged storage, which is a normal phenomenon. The solution can be used after filtration.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
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