A new method to prevent immunohistochemical section drying
In order to prevent the immunohistochemical section from drying, we have repeatedly explored the method of adding the surfactant Tween-20 to prevent dry film in the cleaning buffer. It has been used for more than 2 years and the effect is good. Now introduced as follows.
1. Materials and methods
1.1 Materials
PBS phosphate buffer (0.01 mol/L, pH 7.2-7.4), Tween-20, 500 ml volumetric flask and absorbent paper.
1.2 method
Take 500 ml of prepared PBS phosphate buffer solution into a volumetric flask, add 2 to 3 drops of Tween-20 to the bottle, and mix well. The sections of 10 different tissues were washed according to the immunohistochemical staining procedure in a conventional PBS phosphate buffer, and the sections were placed in the prepared PBS-Tween-20 buffer, and pulled up and down several times to uniformly cover them. On the whole slice, wipe off the excess liquid around with the absorbent paper and put it into the wet box to add the reagent to the next step. In order to prevent the occurrence of edge effects, it is required to wipe from the edge of the tissue <2mm, while processing 10 pairs of photos with crayons and toothpick methods.
2, the results
In the crayon group, there were 2 cases in which the tissue edge was dry during the incubation of the antibody, and the edge effect occurred. However, no edge effect occurred in the Tween-20 group, and the staining was more uniform. The addition of Tween-20 buffer will better prevent the slices from drying out.
3 Discussion
In immunohistochemical staining, slice drying causes non-specific binding of antibodies to tissues, resulting in false positives. Especially at the edge of the tissue, if the antibody is not covered enough, it will easily cause dry film, resulting in non-specific staining, that is, "edge effect" [1]. The common method used in the past is to use crayons and wet boxes. It is cumbersome to use by 5 crayons and needs to be replaced frequently. Most units now use a dedicated immunohistochemical pen. Using a crayon or an immunohistochemical pen can only prevent the added reagent from escaping to the surrounding area. If the tissue is too large, there is no guarantee that the added reagent will completely cover the entire tissue at one time, and other tools (such as toothpicks, etc.) will be needed. The antibody covers all tissues, which increases the complexity of the immunohistochemistry and may increase the likelihood of uneven coverage of the antibody. Tween-20 is a mixture of polyoxyethylene sorbitan monolaurate and a portion of polyoxyethylene bissorbitan monolaurate. Because it has more hydrophilic groups (polyoxyethylene) in its molecule, it is highly hydrophilic, has emulsification, diffusion, solubilization, stability and other properties. It is a commonly used non-ionic detergent and Surfactant, which is harmless to the human body and non-irritating, is commonly used as an emulsifier, a diffusing agent and a stabilizer for pharmaceuticals. Immerse the slice into it to cover the entire slice evenly, as if a liquid film is added to the slice, which will not form a clear boundary between the tissue and the blank area of ​​the slice. After wiping with the absorbent paper at <2mm from the edge of the tissue, buffer The fluid creates a clear boundary around the tissue, and the added reagents are evenly dispersed throughout the tissue, allowing the entire section to be evenly dyed. Due to the surface tension of the buffer, the sealed reagent does not diverge around, and a relatively large amount of buffer can remain on the tissue, which better prevents the occurrence of dry sheets and avoids edge effects. In addition, because Tween-20 is a detergent, tissue sections can be cleaned more cleanly, avoiding the occurrence of non-specific staining. This method has been used in this laboratory for more than 2 years. It has been tested several times without Tween-20 solution. It is found that Tween-20 has no effect on immunohistochemical staining results and can be used with confidence. Moreover, the preparation method of PBS-Tween-20 buffer is simple, and the general laboratory can complete the operation; the price is cheap, the medical cost is saved, and the application is easy to popularize.
references:
[1] Zhou Xiaoge. Problems and countermeasures in immunohistochemical staining process [J]. Journal of Diagnostic Pathology, 2004, 1(4): 211-213.
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