I. ABC method (SP, LSAB, SABC method with the same steps)
ABC (avidin-biotin comeplex) ovalbumin-biotin complex method belongs to affinity immunohistochemistry technique, according to the biological property of high affinity of ovalbumin-biotin, biotin peroxidase is obtained by combining biotin with peroxidase, and then adding ovalbumin and biotin to form ovalbumin-biotin-peroxidase complex. While LSAB, S-P and SABC are using anti-biotin protein chain enzyme instead of ovalbumin, and anti-biotin protein chain enzyme (SA) labeled with peroxidase instead of ABC complex, the steps are the same, but the reaction speed is faster due to the smaller molecular weight of SA and better penetration. Moreover, SA has two binding sites with very high affinity to biotin, and it has no biotin attached to itself, which can link with biotin on secondary antibody, so it is easier to bind with biotin on secondary antibody than ABC, so it is more sensitive, and the complex does not need to be mixed before use, which is simpler and more convenient. ABC is a three-step method, and the secondary antibody is biotinylated IgG (or IgM, IgA, etc.), where the Fab fragment Binding to the first antibody, this requires matching with the first antibody. If the first antibody is a mouse antibody, the second antibody should be anti-mouse (or IgM, IgA, etc.); if the second antibody is a rabbit antibody, the second antibody should be anti-rabbit (or IgM, IgA, etc.), as well as antibodies of sheep, rat, horse and other sources. If a mouse and rabbit universal secondary antibody is used, it is not necessary to divide it when the primary antibody is mouse or rabbit. ABC is 20-40 times more sensitive than PAP because of the high affinity of ovalbumin and biotin. Finally, the peroxidase on the complex reacts with the color development to form a colored precipitate, which can be observed microscopically. The best chromogenic agent for peroxidase is DAB, which forms brown particles insoluble in water and can be dehydrated with alcohol and transparent with xylene; if AEC is used for color development, red precipitate dissolved in water is generated, so it cannot be dehydrated and can only be sealed with water-soluble sealer, which is not good for long-term storage.
Steps.
1.Slice dewaxing to water
2.Block endogenous peroxidase (198 ml methanol or water + 2 ml 30% H2O2) at room
temperature for 20 min. Wash with water.
3, Antigen repair: (1) pH 7.2 TBS buffer washed three times, trypsin digestion 37 ℃, 30 min. Or (2) according to the instructions: distilled water wash, put into antigen repair solution (Ph6.0) within the microwave repair 20 minutes, or electric furnace heating 30 minutes, or autoclave with cap out of gas 3 minutes, rapid cooling; or (3) according to the instructions: no treatment)
4, pH 7.2 TBS buffer wash three times.
5, add normal serum drop by drop 37 ℃, 30 minutes.
6, shake off excess serum, add primary antibody 37 ℃, 30 minutes. Then, add the first antibody for 30 minutes.
7.Wash three times with pH 7.2 TBS buffer.
8, add secondary antibody dropwise, 37 ℃, 30 minutes.
9. Wash three times with pH 7.2 TBS buffer.
10. Add ABC complex dropwise, 37 ℃, 45 min.
11, Wash three times with pH 7.2 TBS buffer.
12.DAB color development for 3-10 min.
13.Water wash.
14, Hematoxylin light staining, bluing, dehydration, transparency, sealing.
II. PAP method
Steps.
1.Section dewaxing to water
2, blocking endogenous peroxidase (198 ml methanol or water + 2 ml 30% H2O2) at room
temperature for 20 min. Wash with water.
3, Antigen repair: (1) pH 7.2 TBS buffer washed three times, trypsin digestion 37 ℃, 30 min. Or (2) according to the instructions: distilled water wash, put into antigen repair solution (Ph6.0) within the microwave repair 20 minutes, or electric furnace heating 30 minutes, or autoclave with cap out of gas 3 minutes, rapid cooling; or (3) according to the instructions: no treatment)
4, pH 7.2 TBS buffer wash three times.
5, add normal serum drop by drop 37 ℃, 30 minutes.
6, shake off excess serum, add specific antibody drop by drop 37 ℃ for 30-60 minutes or 4 ℃ overnight, place in wet box.
7, PBS wash.
8.Add secondary antibody drop by drop, 37 ℃, 30 minutes.
9, pH 7.2 TBS buffer washed three times.
10, Add PAP antibody 37 ℃ for 30 minutes.
11, pH7.2 TBS buffer wash three times, DAB color development for 3-10 minutes.
12.Water wash.
13, Hematoxylin light staining, bluing, dehydration, transparent, sealing.
III. APAAP method
APAAP: It belongs to the non-labeled antibody method, through the anti-alkaline phosphatase antibody with high specificity and adding a large amount of alkaline phosphatase to the anti-enzyme antibody, so that the alkaline phosphatase is fully bound to the anti-enzyme antibody to form a soluble APAAP complex, the commonly used color developer is BCIP/NBT, solid red or solid blue. Features will not react with endogenous peroxidase, so the background is cleaner, especially suitable for tissues with more endogenous peroxidase, such as liver and kidney.
Steps.
1, section dewaxing to water (frozen sections or cell smears soaked in PBS for 3 minutes, no antigen repair)
2, block endogenous peroxidase (198 ml methanol or water + 2 ml 30% H2O2) at room
temperature for 20 min. Wash with water.
3, Antigen repair: (1) pH 7.2 TBS buffer washed three times, trypsin digestion 37 ℃, 30 min. Or (2) according to the instructions: distilled water wash, put into antigen repair solution (Ph6.0) within the microwave repair 20 minutes, or electric furnace heating 30 minutes, or autoclave with cap out of gas 3 minutes, rapid cooling; or (3) according to the instructions: no treatment)
4, pH 7.2 TBS buffer wash three times.
5, add normal serum drop by drop 37 ℃, 30 minutes.
6, shake off excess serum, appropriate dilution of specific antibodies, 37 ℃ for 60 minutes, or 4 ℃ overnight.
7, TBS or PBS (pH 7.2) wash 3 times.
8, Bridge antibody, 37°C for 40 min or 1 hr at room temperature.
9, TBS or PBS (pH 7.2) wash 3 times.
10, Dropwise addition of appropriately diluted APAAP complex, 37°C for 30 min.
11, Wash 3 times with TBS or PBS (pH 7.2).
12.Add AKP substrate solution drop by drop, 37℃ for 15–30 minutes, rinse with water after the positive result appears clear.
13.Re-staining (nuclear solid red or hematoxylin or methyl green) for 1-2 minutes, after regular rinsing, glycerol gelatin seal the film.
IV. LDP (labelled dextran polymer) method (Envision two-step method)
Enzyme-labeled polymer method, the application of enzyme-labeled polymer technology, such as EnVison detection system, the use of horseradish peroxidase or alkaline phosphatase labeled on the glucose polymer, and then ligated with secondary antibody, forming EnVison secondary antibody; like the PowerVision detection system, the use of horseradish peroxidase or alkaline phosphatase labeled on the amino acid fragment. Since more than 20 sites on the polymer can bind to the primary antibody and the number of enzymes on the polymer is higher, it is more sensitive than methods such as ABC and LSAB. Most importantly, since there is no biotin present on the secondary antibody of the LDP system, there is no cross-reaction with endogenous biotin binding as seen in ABC, S-P and other methods, reducing non-specific coloration and a cleaner background.
Steps.
1, section dewaxing to water
2, 0.3% H2O2 methanol solution: block endogenous peroxidase (198 ml methanol or water + 2 ml 30% H2O2) for 20 min at room temperature. Tap water wash.
3. Antigen repair: (1) pH 7.2 TBS buffer wash three times, trypsin digestion 37 ℃, 30 min. Or (2) according to the instructions: distilled water wash, put into antigen repair solution (Ph6.0) within the microwave repair 20 minutes, or electric furnace heating 30 minutes, or autoclave with cap out of gas 3 minutes, rapid cooling; or (3) according to the instructions: no treatment)
4, pH 7.2 PBS buffer wash three times.
5, add normal serum drop by drop 37 ℃, 20 minutes.
6, shake off excess serum, add primary antibody, 37 ℃, 30 minutes.
7.Wash three times with pH 7.2 TBS buffer.
8.Add Envision secondary antibody dropwise, 37 ℃, 30 minutes.
9, pH 7.2 TBS buffer washed three times.
10.DAB color development for 3-10 minutes.
11.Water wash.
12, Hematoxylin light staining, bluing, dehydration, transparency, sealing.
V. Double staining method
1.Section dewaxing to water
2, Block endogenous peroxidase (198 ml methanol or water + 2 ml 30% H2O2) at room temperature
20 min. Wash with water.
3, Antigen repair: (1) pH 7.2 TBS buffer washed three times, trypsin digestion 37 ℃, 30 min. Or (2) according to the instructions: distilled water wash, put into antigen repair solution (Ph6.0) within the microwave repair 20 minutes, or electric furnace heating 30 minutes, or autoclave with cap out of gas 3 minutes, rapid cooling; or (3) according to the instructions: no treatment)
4, pH 7.2 TBS buffer wash three times.
5.Drop add normal serum 37 ℃, 30 minutes. (can be used without)
6, shake off excess serum, add primary antibody 37 ℃, 30 minutes.
7, pH 7.2 TBS buffer washed three times.
8.Add biotinylated secondary antibody dropwise, 37 ℃, 30 minutes.
9. Wash three times with pH 7.2 TBS buffer.
10. Add ABC or S-P complex (horseradish peroxidase) dropwise, 37 ℃, 45 min.
11. Wash three times with pH 7.2 TBS buffer.
12, DAB (brown) or AEC (red) color development for 3~10 min.
13, pH 7.2 TBS buffer washed three times.
14.Add normal serum dropwise at 37 ℃ for 30 minutes. (Can be used without)
15, shake off the excess serum, add the second primary antibody 37 ℃, 30 minutes.
16, pH 7.2 TBS buffer washed three times.
17, add biotinylated secondary antibody dropwise, 37 ℃, 30 minutes.
18. Wash three times with pH 7.2 TBS buffer.
19.Add alkaline phosphatase complex dropwise, 37 ℃, 30 min.
20, Wash three times with pH 7.2 TBS buffer.
21, BCIP/NBT color development (blue)
22.Water wash
23, Methyl green re-staining, dehydration, transparent, and sealed.
If choose horseradish peroxidase can choose DAB (brown) color development, in step 21 with horseradish peroxidase complex, step 23 with AEC (red) color development, but it should be noted that AEC can not be stored for a long time. If you choose another for alkaline phosphatase can be used BCI/NBT, AP-Red, AP-Orange, with BCI/NBT color development is best.
VI. Immunogold method (IGS)
(1) The sections are routinely dewaxed into water.
(2) Digest with 0.1% trypsin solution at 37℃ for 10-30 minutes.
(3) Double distilled water wash 2 times.
(4) Wash with 0.05 mol/L TBS PH7.4 for 10 minutes.
(5) 1% ovalbumin closure for 10 min.
(6) Add specific primary antibody dropwise for 2 hours at room temperature or overnight at 4°C.
(7) Wash 3 times with 0.5 mol/L TBS PH7.4.
(8) 1% ovalbumin closure for 10 min.
(9) Dilute gold standard antibody for 45 min at 37°C.
(9) 0.05 mol/L TBS PH7.4 for 3 times.
(10) Double distilled water wash 2 times.
(11) 1% Haldol for 10 min.
(12) Double distilled water wash 3 times.
(13) Re-staining, glycerol sealing and observation.
Results: The colloidal gold binding site is red.
VII. Immunogold and silver method (IGSS)
(1) The sections were routinely dewaxed into water.
(2) Tissue sections fixed by mercury-containing fixative should be de-mercuryed by 0.5% iodine alcohol, then de-iodized in 0.5% thiosulfate aqueous solution, rinsed with running water, washed with double distilled water, and washed with 0.05 mol/L TBS PH7.4 solution.
(3) Digestion with 0.1% trypsin solution, 37℃ for 10-30 minutes.
(4) Wash with 0.05 mol/L TBS PH7.4.
(5) Closure with 1% ovalbumin for 10 min.
(6) Titrate with appropriate dilution of specific antibody for 1-2 hours at 37°C or overnight at 4°C.
(7) Wash 3 times with 0.05 mol/L TBS PH7.4 solution.
(8) 1% ovalbumin closure for 10 minutes.
(9) Titrate with appropriate dilution of gold-labeled antibody at room temperature for 45 min at 37°C.
(10) Wash 3 times with 0.05 mol/L TBS PH7.2.
(11) Wash 2 times with distilled water and 2 times with double distilled water.
(12) Put into silver developing solution for 3-5 minutes, avoid light during the reaction.
(13) Distilled water wash.
(14) Tap water rinse, hematoxylin light re-staining.
(15) Dehydration, transparency, sealing and observation.
Results: The specific antibody binding site is gray-black particles, the background is clear gray, the nucleus is light blue.
Reagent preparation.
Silver developer.
1, silver nitrate developer.
(1) 10% aqueous gum Arabic solution 60ml, citric acid buffer pH 3.510ml
(2) 1.7g of hydroquinone plus 30ml of double distilled water.
(3) 40mg of silver nitrate plus 2ml of double-distilled water.
(1) and (2) two liquids are completely dissolved and mixed, before use add (3), dark place to develop.
2. 0.05 mol/L TBS (pH 7.4).
Tris 12.1g,
NaCl 17.5g,
Stir well to dissolve, adjust the pH to 7.4 with concentrated HCl, then add double distilled water to 2000ml.