Permeabilization reagent, also known as permeabilization solution, usually achieves cell membrane permeabilization by using organic solvents such as methanol and acetone, or detergents such as Triton X-100 and Saponin.
Organic solvents like methanol or acetone can, on the 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 play a fixing role. Their advantages lie in simple operation-one-step treatment can achieve both fixation and permeabilization simultaneously. However, their disadvantages are that membrane proteins will also be dissolved, and the denaturation of some proteins is not conducive to subsequent detection. Therefore, organic solvents are relatively less used and only applicable to some rough detection requirements. Triton X-100 is a commonly used permeabilization reagent that can permeabilize cell membranes and nuclear membranes. Its mechanism of action is the non-specific dissolution of cell membranes. A drawback is that it is not conducive to the detection of membrane proteins. Nevertheless, after cross-linking and fixation with paraformaldehyde, a considerable part of membrane proteins will be cross-linked and fixed, 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 by flow cytometry. The disadvantages include poor permeabilization effect on cells with low cholesterol content, a weaker permeabilization effect compared with Triton X-100 and organic solvents, and the inability to permeabilize nuclear membranes and mitochondrial membranes with very low cholesterol content. For the detection of lectins, the permeabilization solution containing non-specific detergents such as Triton X-100 has a significantly better effect than the one mainly containing Saponin.
PBS-Saponin Solution (Permeabilization Reagent, 0.1%), also called immunostaining permeabilization solution, is mainly composed of 0.01M PBS (phosphate-buffered saline, pH 7.4) and 0.1% 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 such as antigens and nucleic acids, facilitating the entry of antibodies, probes or labels into cells, thus ensuring the detection effect 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, and is particularly suitable for membrane protein detection. This product does not dissolve cell membranes and will not affect light scattering during flow cytometry detection. 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 directly applied 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 fixation and washing steps, add 50-100 μl of PBS-Saponin Solution dropwise to each sample, or completely immerse the sections in a staining jar for permeabilization.
2. For cell samples: After 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 according to this proportion.
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 10-20 minutes to complete permeabilization. For samples that are difficult to permeabilize or when thorough permeabilization is required, the incubation time can be extended to 20-40 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 the detection of lectins, PBS-Triton Solution (Permeabilization Reagent, 0.1%) or PBS-Triton Solution (Permeabilization Reagent, 0.3%) is recommended.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
6. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
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