Paraffin section immunohistochemical staining steps

1. Handling of slides: In the process of antigen repair, due to the influence of many factors such as high temperature, high pressure, radiation, etc., it is easy to cause off-chip. In order to ensure the normal operation of the test, you can use ZLI-9001 APES, ZLI-9003 HistogripTM or ZLI-9005 Poly-L-Lysine provided by our company to process the cleaned slides. The specific methods are as follows: 1.1 APES: currently used and configured. Put the washed slides in APES diluted with 1:50 acetone, and stay for 20 ~ 30 seconds, take out for a while, then put in pure acetone solution or distilled water to rinse off the unbound APES, and put it in a fume hood Allow to dry. When using this slide to remove slides, it should be noted that the tissue should be in one step, and the presence of air bubbles should be minimized to avoid affecting the staining result. 1.2 HistogripTM: Put the washed slides into Histogrip solution diluted with 1:50 acetone, stay for 1 ~ 2 minutes, then quickly rinse three times with double distilled water, dry at room temperature or bake in a 60oC oven for one hour, box spare. 1.3 Poly-L-Lysine: Put the washed and dried slides into a polylysine solution diluted with 1:10 deionized water, soak for 5 minutes, bake in a 60oC oven for one hour or dry at room temperature overnight . Boxed for future use. The appliances used in the test are all non-glass products. 2. Common enzyme digestion: 2.1 Trypsin: The general concentration is 0.05% ~ 0.1%, the digestion time is 37 ℃, 10 ~ 40 minutes, mainly used for the display of intracellular antigens. 2.2 Pepsin: generally used at a concentration of 0.4%, digestion time is 37 ℃, 30 ~ 180 minutes, mainly used for the display of interstitial antigens, such as: Laminin (lamin), Collagen IV (type IV collagen) and so on. The digestion time of g / ml saponin solution is 30 minutes at room temperature. 2.3 Saponin: generally used at a concentration of 2 to 10 3. Antigen heat repair: According to the specific conditions of the laboratory, microwave antigen repair, autoclave antigen repair or water bath high temperature antigen repair can be selected. Various buffers can be used for antigen hot repair, such as TBS, PBS, heavy metal salt solution, etc., but experiments have shown that 0.01M citrate buffer (pH 6.0) is the best. Please use ZLI-9064® citrate buffer (powder) provided by our company to prepare, take a packet of the powder and dissolve it in 1000ml of distilled water, mix well, its pH value is 6.0 0.1, such as the pH caused by the distilled water itself Value deviation, please adjust by yourself. 3.1 Paraffin section microwave oven antigen repair operation method: After the section is dewaxed to water, treated with 3% H2O2 for 10 minutes, and washed with distilled water for 2 minutes × 3. Place the slices in a container filled with citrate buffer (working fluid) and heat it in a microwave oven to keep the temperature of the liquid in the container between 92 ° C and 98 ° C for 10 to 15 minutes (note: whether it is used (Medical or household microwave ovens, please set the conditions according to the specific model, and must meet the temperature and time requirements in the above steps). Take out the container and cool it at room temperature for 10 ~ 20 minutes (Note: Do not take the slice out of the buffer and cool it, so that the protein can restore the original spatial configuration). Wash with PBS, followed by immunohistochemical staining steps. 3.2 Paraffin section high-pressure antigen repair operation method: section dewaxed to water. Inject 1500ml ~ 3000ml of citrate buffer solution (working fluid) into a stainless steel pressure cooker and heat to boiling. Place the slice on a metal rack, put it in the pot, make the slice below the liquid level, and cover the pot pressure valve. When the pressure cooker starts to spray slowly (after about 5 to 6 minutes of heating), count the time for 1 to 2 minutes, then remove the end of the pressure cooker from the heat source, cool the water to room temperature, remove the air valve, open the lid, take out the slices, and wash with distilled water After that, wash with PBS for 2 minutes × 3, followed by the immunohistochemical staining step. 3.3 Paraffin section electric stove boiled antigen repair operation method: After dewaxing the section to water, put it in a container containing citrate buffer (working fluid), and place this container in a large vessel containing a certain amount of tap water , Heat and boil on the electric furnace, start the timer for 15-20 minutes from the temperature of the small container reaching 92 ℃ ~ 98 ℃, and then leave the electric furnace, cool at room temperature for 20-30 minutes, rinse with distilled water, wash with PBS, then follow the immunohistochemical staining step. 4. Immunohistochemical staining steps: (using the SP kit of ZYMED company in the United States as an example) Paraffin sections are dewaxed to water. Incubate 3% H2O2 at room temperature for 5 ~ 10 minutes to eliminate the activity of endogenous peroxidase. Rinse with distilled water and soak in PBS for 5 minutes. (If antigen repair is needed, it can be done after this step). 5-10% normal goat serum (diluted in PBS) was blocked and incubated at room temperature for 10 minutes. Drain the serum, do not wash, add the primary antibody or primary antibody working solution diluted in appropriate proportion, and incubate at 37 ℃ for 1 ~ 2 hours or 4 ℃ overnight. Rinse with PBS, 5 minutes × 3 times. Add the biotin-labeled secondary antibody (1% BSA-PBS dilution) diluted in appropriate proportion and incubate at 37 ℃ for 10 ~ 30 minutes; or add the second-generation biotin-labeled secondary antibody working solution at 37 ℃ or room temperature for 10-30 minute. Rinse with PBS, 5 minutes × 3 times. Add the appropriate ratio of horseradish enzyme-labeled streptavidin (diluted in PBS) and incubate at 37 ℃ for 10-30 minutes; or the second-generation horseradish enzyme-labeled streptavidin working solution, incubate at 37 ℃ or room temperature for 10-30 minute. Rinse with PBS, 5 minutes × 3 times. Color developer (DAB or AEC). Rinse thoroughly with tap water, counterstain and seal. Immunohistochemical staining of frozen sections. After standing at room temperature for 30 minutes, fix it in acetone at 4 ° C for 10 minutes, wash with PBS for 5 minutes × 3. Incubate with 3% hydrogen peroxide for 5-10 minutes to eliminate endogenous peroxidase activity. Wash with PBS, 5 minutes × 2. The elimination method of frozen section 4 ~ 8 immunohistochemical non-specific staining 1. The main factors of non-specific staining The mechanism of non-specific staining of tissue is very complicated. The reasons for its generation can be divided into the following points: (1) Part of fluorescence The protein does not bind to the protein, forming polymers and derivatives, which cannot be removed by dialysis. (2) Serum proteins other than antibodies combine with fluorescein to form fluorescein urea protein, which can be combined with tissue components. (3) In addition to the antigens to be examined, there may be generic antigens (such as Forssman's antigen) in the tissue, which can be combined with corresponding antibodies other than specific antigens in the tissue. (4) It is difficult to purify antigenic substances from tissues, so the prepared immune serum is often mixed with some antibodies against other tissue components, which is easy to confuse. (5) There are too many fluorescein molecules labeled on the antibody molecule. Such excessively labeled antibody molecules carry too many anions and can be adsorbed on normal tissues and present non-specific staining. (6) Impure fluorescein, improperly fixed specimens, etc. Second, the method to eliminate non-specific staining The method to eliminate non-specific staining of fluorescent antibodies should be based on the reason for the appropriate method, the common methods are as follows: (1) animal organ powder absorption method commonly used liver powder (pig, large White rat or mouse), followed by bone marrow meal, rat brain meal and chicken embryo meal. Add 50-100 mg of liver powder to each ml of fluorescent antibody, mix well in a centrifuge tube, shake at room temperature for 2h, overnight at 4 ° C, stir for another 10min, high-speed centrifuge (3000 ~ 15000r / min) for 30min, 1-2 times After that, the supernatant can be used. Absorption should generally be performed before use, and the fluorescent antibody after absorption should not be stored in the refrigerator for more than 2 weeks. Staining should be compared before and after absorption. During absorption, tissue dry powder can be soaked with buffered saline, centrifuged (3000 ~ 15000r / min) for 30min, the supernatant is removed, and fluorescent antibody is added for absorption, so as not to consume too much antibody. Absorption of liver powder or fresh cells is a non-specific method of elimination, which has an adsorption effect on fluorescent pigments and proteins of fluorescent antibodies. For example, when examining virus antigens in tissues, they can also be absorbed by the same tissue dry powder or homogenate sediment. Absorption of organ liver powder will result in more loss of fluorescent antibodies. If the 20% saline homogenate of the tissue is washed with saline 2 to 3 times according to the method of Hiramotos et al., Centrifuged at 12000r / min for 10 minutes, and the precipitate Absorbing its fluorescent antibody can achieve the goal completely. Kyoji Fang Jiu Shi believes that such absorption will have almost no loss on the fluorescent antibody. They usually use this method, and the effect is very good. It is placed for about a week after absorption. [Methods of making liver powder] (1) Bleed and kill a few mice or rats, remove the liver, wash it with physiological saline 2 to 3 times, remove the blood, and strip the fat of the connective tissue on the surface. (2) Crushed, washed repeatedly with normal saline until no blood color, then add a little more normal saline, and make homogenate with a tissue masher or homogenizer. (3) Put the liver homogenate into a centrifuge tube (about 1/3), exchange it with 2 to 3 times the amount of normal saline and acetone and wash it three times repeatedly, until the supernatant is bloodless, centrifuge at 2000r / min every time After precipitation for 15 min, the supernatant was removed again. (4) Finally, the liver slurry was washed with acetone, then filtered with a Buchner funnel, or centrifuged to precipitate, and the precipitate was spread on a clean glass plate and baked at 37 ° C (overnight). (5) Grind it thoroughly in a mortar, sieve through a 120-mesh copper sieve, pack it separately, seal it, and store it at low temperature. (B) Dialysis method Fluorescein such as FITC molecules can pass through the semi-permeable membrane, but protein macromolecules are not permeable, and fluorescein that is not bound to the protein can be dialyzed and removed. (1) Put the labeled fluorescent protein solution into a dialysis bag or cellophane bag, leaving a slight gap in the liquid surface, and tie it tightly. (2) Immerse in 0.02mol / pH 7.1 ~ 7.4 PBS (suspended in PBS about 50-100 times larger than the volume of the marker), dialyze at 4 ℃, change PBS 3 to 4 times a day, about 5 to 7 Days, there is no fluorescence in the dialysate (under the fluorescent light source). (3) Dextran gel G-50 column chromatography can be used to remove free fluorescein. 2 × 46cm column chromatography can be used. For detailed methods, please refer to Chapter 2. Add 15-18ml of fluorescent antibody (add 5% -10% of the bed volume), slowly infiltrate into the column. When all of the column is about to be added, add a little PBS, close the lower mouth, and stay for 30-40min to make free fluorescence Fully enter the fine sieve hole, then turn on the elution bottle and start dropping the eluent. After adding a certain amount of eluent, the fluorescent antibody will move downward, and gradually draw a clear boundary between the free fluorescein remaining at the upper end. With the continuous addition of a large amount of eluent, the separation distance between the two becomes larger and larger. The fluorescent antibody is flowed out first, collected in three parts: front, middle, and back, and the F / P ratio is measured. Those who pass the test are combined, concentrated, and packed. The eluent is determined by 20% sulfosalicylic acid to determine the protein (precipitation reaction occurs), and the elution is continued. The free fluorescein is eluted successively. When there is no protein and fluorescein in the eluent, the chromatography column is Reusable. If it is used to remove the free fluorescein and ammonium sulfate and other salts in the fluorescent antibody, it can be dialyzed overnight before passing through the column, otherwise, NH4 + is too concentrated, and NH4 + appears when the protein is not completely eluted, thus affecting purification and protein recovery In general, when protein appears in the eluent, it is collected, and then SO4 ++ appears (check with 1% BaCl2 for white precipitation). Finally, NH4 +, (the yellow-brown precipitate is checked with Nessler's reagent), and can be reused after the eluent is free of SO4 ++ and NH4 +. If only a small amount of fluorescent antibody is used, a 1 × 20 cm column chromatography column can be used, and 2 g of Sephadex G-50 is packed into the column to filter 2 to 3.5 ml of fluorescent antibody. (4) DEAE-cellulose column chromatography method to label too much or too little fluorescein antibody molecules can be removed by DEAE-cellulose column chromatography. The method is as follows: DEAE-cellulose column packing, elution and regeneration methods are the same as those for purifying IgG. The amount of DEAE-cellulose required for column packing is preferably based on the exchange of 20-50 mg of labeled protein per gram of dry weight. The commonly used gradient elution method is as follows: (1) The chromatography column is equilibrated with 0.01mol / L, pH7.2PB. After the label is applied to the column, it is first eluted with 0.01mol / L, pH7.2PB, washing out colorless or light green Liquid, the amount of eluent (multiplied by 3 per gradient according to the bed volume), and then eluted and collected according to the following various ionic strength eluents: 0.01mol / L, pH7.2PBS (0.05mol / L NaCl) ... ... eluting section 1. 0.01mol / L, pH7.2PBS (0.01mol / L NaCl) ... Elution part 2. 0.01mol / L, pH7.2PBS (0.02mol / L NaCl) ... Elution part 3. The three parts of the collected solution (5ml per tube) were measured for their F / P ratio. 0.05mol / L NaCl pH7.2PB eluent 280nm optical density peak tube was combined, concentrated and stored for later use. Because this part of non-specific staining has the least fluorescence, it is a good fluorescent antibody. The other two parts can be discarded. (2) The excessively labeled protein adsorbed on the column can continue to increase the concentration of NaCl to 2.0 mol / L after elution. The amount of labeled antibody after DEAE-cellulose chromatography is generally about 50% lost, so some less demanding antibodies, such as anti-bacterial fluorescent antibodies, do not have to be treated in this way, and the dilution method for determining the staining titer can be used Remove non-specific stains. (Five) Fluorescent antibody dilution method first determines the titers of fluorescent antibody specific staining and non-specific staining. If the two titers differ greatly, the fluorescent antibody can be diluted to a critical concentration to make the specific staining positive. To keep non-specific staining negative, the dilution method and staining titer determination method are the same. (6) Purification of antigen method Purification of single-component antigens by various methods is the most important condition for the production of monovalent specific antibodies. Modern immunochemical technology (immunoabsorption method) and column chromatography provide a great possibility, you can refer to the relevant monographs. (7) Purified antibody method --- immunoabsorption method such as purification method of anti-IgA serum --- immunoabsorption method. If the secretion type IgA (SIgA) antigen purity is not high, the prepared antiserum often cross-reacts with IgG, so it needs to be absorbed. It is often purified by using purified human IgG glutaraldehyde polymer. The method is as follows: 1. Preparation of human IgG polymer In 5 ml of 0.1 mol / L pH 7.0 phosphate buffer solution containing 40 mg / ml of human IgG, add 1 ml of 2.5% glutaraldehyde solution, stir while adding, 5 minutes will appear turbid, and large After leaving the gel for 30 minutes, the gel was ground finely with a mortar, followed by repeated washing with 1.0 mol / L pH 7.0 phosphate buffer solution three times, and the final addition of distilled water to 20 ml was the human IgG polymer suspension. 2. Immunoabsorption method: Add the anti-SIgG serum to be absorbed into the required amount of IgG polymer suspension, stir at room temperature for 60 min, centrifuge to precipitate, and the supernatant is diluted by 1 fold purified anti-SIgA serum. If IgG polymer is used for small amount of fractional absorption, its effect is better. (Eight) Evans blue (Evans blue) staining method with 0.01% Evans blue 0.01mol / L pH7.2PBS diluted fluorescent antibody, can stain background cells and tissues, showing red fluorescence, and specific yellow-green fluorescence formation The sharp contrast reduces non-specific fluorescence and is suitable for routine applications. Evan's blue is generally formulated into a 1% solution, stored at 4 ° C, and diluted to 0.01% before use to release fluorescent antibodies. In addition, non-specific staining can be eliminated by digesting tissue sections with trypsin or using 10% bovine serum protein blocking method to improve specific staining. Source: Biotechnology Forum

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