Immunohistochemistry experiment

Stripping is a problem often encountered in immunohistochemistry (especially frozen sections of brain tissue). In general, there are several reasons for stripping:

1. Tissue fixation, dehydration, and insufficient transparency

2. Tissue section is too thick

3. Tissue section has fold

4. Excessive thermal antigen retrieval (high pressure, microwave, boiled) treatment or high pH of antigen retrieval fluid

5. During the operation, the flushing method is incorrect, etc.

In the process of actual operation, in addition to paying attention to the above points, the application of anti-offset film is very necessary. The commercially available anti-offset slides are generally positively charged slides, and the quality of different brands is different. Even the better PREMIER slides are sometimes unsatisfactory and have a lot of fakes. Therefore, labs often need to handle the anti-off slides themselves. The steps are as follows:

1. Acid treatment of slides

The sub-strong acid cleaning solution (120 g of potassium dichromate, 200 ml of concentrated sulfuric acid, 1000 ml of distilled water) used in the laboratory was used to soak the slide for 24 hours, rinsed thoroughly with running water, washed three times with distilled water, and immersed in 95% ethanol for 12 hours. Remove and dry in a drying oven or air dry.

2. Use of adhesives

There are three commonly used adhesives: gelatin-potassium chrome sulfate, APES (3-aminopropyl-ethoxysilane), poly-Lysine (poly-1-lysine)

(1) Gelatin - Potassium sulphate potassium, this method is very simple and practical, not only for traditional dyeing, but also for immunohistochemistry, in situ hybridization, etc. Currently, the adhesive can be routinely used in most laboratories. The ointment of this method is that in an alkaline environment with a pH higher than 8.0 (such as TE antigen repair solution using PH9.0), the temperature is increased to 80 degrees or more, and the slice is easy to fall off. Therefore, PH6 should be used when selecting such an adhesive. 0 sodium citrate antigen repair solution.

Preparation method: 5 g of gelatin is dissolved in 1 L of hot distilled water. After cooling, 0.5 g of potassium sulphate potassium is added and stirred and dissolved. The slide is immersed in the glass for 30 minutes, taken out and naturally dried, and then immersed for another 30 minutes. . If the effect is not sufficient, the concentration can be doubled, that is, 10 g of gelatin + 1 g of potassium sulphate / L. (higher concentrations are not tried in this laboratory)

(2) APES (3-aminopropyl-ethoxysilane) should be diluted with acetone, and acetone has the effect of causing cirrhosis. Pay attention to personal protection. This method compensates for the problem of partial high temperature alkaline solution stripping. In addition, there is a saying that if there is a phenomenon of peeling off the film that has been attached, it can be soaked for 3 minutes in this method, and dried for the next step, but the results verified by the laboratory are not good.

Preparation method: APES is diluted with acetone (APES: acetone = 1:50), the washed slide is immersed in the solution for 20 seconds, the excess solution is taken out, and then immersed in pure acetone or distilled water to wash away unbound APES. The dry box is ready for use. (Note: This solution needs to be used now, so try to process as many slides as possible in one batch to make the most of it.)

(3) Poly-Lysine (poly-1 - lysine), which is quite expensive. However, if high pressure alkaline repair is performed, the overall effect may be optimal. Ordinary histochemical experiments and immunohistochemical acid repair (in most cases) are not necessary for routine use. Polylysine generally has a molecular weight of 70,000 to 150,000, 150,000 to 300,000 and >300,000. The greater the molecular weight, the stronger the adhesion, but the relative dissolution is more difficult.

Preparation method: Dissolve polylysine in double distilled water at a concentration of 0.1 mg/ml, and leave the washed slide in the solution for 5 minutes (the increase time does not improve the adhesion effect), and naturally dry and pack the cartridge. . If you want to save the reagent, you can use the direct coating method, pipette the solution to the area on the slide that requires the patch, and control the area size at will. The general dosage is controlled at 20μl/cm2. (Note: 1. The immersion method generally does not exceed 100 sheets per 100ml of polylysine solution. Excessive coating will reduce the adhesion. 2. Polylysine solution is stored at 4 ° C, stable within one year. 3. The polylysine solution taken out of the refrigerator should be returned to room temperature before use.

Related Links:

• Immunohistochemistry
• Immunofluorescence
• Embedding slice