1. Treatment of slides:
    In the process of antigen retrieval, due to many factors such as high temperature, high pressure, radiation, etc., it is easy to cause stripping. In order to ensure the normal operation of the test, the cleaned slides can be treated with several reagents such as ZLI-9001 APES, ZLI-9003 HistogripTM or ZLI-9005 Poly-L-Lysine provided by our company. The specific method is as follows:
1.1 APES: Available now. Place the washed slide into APES diluted with acetone at a ratio of 1:50, hold for 20~30 seconds, take off for a while, then put it into pure acetone solution or distilled water to remove unbound APES, and put it in the fume hood. Dry it. When using this slide, you should pay attention to the organization in one step, and try to reduce the presence of bubbles, so as not to affect the dyeing results.
1.2 HistogripTM: Place the washed slides in Histogrip solution diluted 1:50 in acetone, leave for 1 to 2 minutes, then quickly wash three times with double distilled water, dry at room temperature or oven for 60 hours, and pack for one hour. spare.
1.3 Poly-L-Lysine: Place the washed and dried slides in a polylysine solution diluted 1:10 in deionized water, soak for 5 minutes, bake in a 60oC oven for one hour or dry at room temperature overnight. . Pack the box for use. The instruments used in the tests were all non-glass products.
2, commonly used enzyme digestion:
2.1 Trypsin: The concentration is generally 0.05%~0.1%, the digestion time is 37°C, 10~40 minutes, which is mainly used for the display of intracellular antigens.
2.2 pepsin: generally used concentration of 0.4%, digestion time is 37 ° C, 30 ~ 180 minutes, mainly used for the display of interstitial antigens, such as: Laminin (layer mucin), Collagen IV (type IV collagen).
 2.3 Saponin: Saponin solution with a concentration of 2~10g/ml is generally used, and the digestion time is 30 minutes at room temperature.
3. Antigen heat repair:
 Microwave oven antigen retrieval, autoclave antigen retrieval or water bath high temperature antigen retrieval can be selected according to the specific conditions of the laboratory. Antigen heat repair can use various buffers, such as TBS, PBS, heavy metal salt solution, etc., but experiments have shown that 0.01M citrate buffer (pH 6.0) works best. Please use ZLI-9064 citrate buffer (powder) provided by our company. Take one pack of this powder and dissolve it in 1000ml of distilled water and mix it. The pH value is 6.0  0.1, such as the pH caused by distilled water itself. Value deviation, please adjust it yourself.
3.1 paraffin section microwave oven antigen repair operation method: slice dewaxing to water, 3% H2O2 treatment for 10 minutes, distilled water for 2 minutes × 3. Place the slice in a container containing the citrate buffer (working fluid) and heat it in the microwave to keep the temperature of the liquid in the container between 92 ° C and 98 ° C for 10 to 15 minutes (note: use For medical or domestic microwave ovens, please set the conditions according to the specific model, and must meet the temperature and time requirements in the above steps). Remove the container and let it cool for 10 to 20 minutes at room temperature (Note: Do not remove the slice from the buffer to allow the protein to restore its original spatial configuration). Wash with PBS and follow the immunohistochemical staining step.
3.2 paraffin section high pressure antigen repair operation method: slice dewaxing to water. Fill 1500ml~3000ml of citrate buffer (working fluid) into a stainless steel pressure cooker and heat to boiling. The slice is placed on a metal frame and placed in a pot so that the slice is below the liquid level and the pan is pressed. When the pressure cooker starts to slowly spray (about 5~6 minutes after heating), count for 1~2 minutes, then remove the pressure cooker from the heat source, chill the water to room temperature, remove the gas valve, open the lid, take out the slices, wash the water with distilled water. After that, the PBS was washed for 2 minutes × 3, followed by an immunohistochemical staining step.
 3.3 Paraffin section electric furnace boiling antigen repair operation method: After the wax is dewaxed to water, it is placed in a container containing citrate buffer (working fluid), and the container is placed in a large vessel containing a certain amount of tap water. Heating and boiling on the electric furnace, starting from the temperature of the small container reaching 92 ° C ~ 98 ° C for 15 to 20 minutes, then leaving the electric furnace, cooling at room temperature for 20 to 30 minutes, washing with distilled water, washing with PBS, followed by immunohistochemical staining step.
4. Immunohistochemical staining steps:
 (Taking the US ZYMED SP kit as an example)
 4.1 Paraffin sections are dewaxed to water.
 4.2 3% H2O2 was incubated for 5-10 minutes at room temperature to eliminate endogenous peroxidase activity.
 4.3 Rinse with distilled water and soak for 5 minutes in PBS (if antigen retrieval is required, it can be done after this step).
 4.4 5~10% normal goat serum (diluted in PBS) was blocked and incubated for 10 minutes at room temperature. Drain the serum, do not wash, add the appropriate dilution of the primary antibody or primary antibody working solution, incubate at 37 ° C for 1 to 2 hours or 4 ° C overnight.
 4.5 Wash with PBS for 5 minutes x 3 times.
 4.6 Add appropriate dilution of biotin-labeled secondary antibody (diluted with 1% BSA-PBS), incubate at 37 °C for 10 to 30 minutes; or add second-generation biotin-labeled secondary antibody working solution, incubate at 37 °C or room temperature for 10~ 30 minutes.
 4.7 PBS rinse, 5 minutes × 3 times.
 4.8 Add the appropriate ratio of horseradish-labeled streptavidin (diluted in PBS), incubate at 37 ° C for 10 to 30 minutes; or second-generation horseradish-labeled streptavidin working solution, incubate at 37 ° C or room temperature 10 ~ 30 minutes.
 4.9 PBS rinse, 5 minutes × 3 times.
 4.10 developer color development (DAB or AEC).
 4.11 The tap water is fully rinsed, counterstained and sealed.
 
Frozen section immunohistochemical staining step
Frozen sections 4~8m, placed at room temperature for 30 minutes, fixed in acetone at 4 ° C for 10 minutes, washed with PBS, 5 minutes × 3. Incubate with 3% hydrogen peroxide for 5-10 minutes to eliminate endogenous peroxidase activity. Wash with PBS for 5 minutes x 2.
The immunohistochemical staining procedure is followed.


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