Polypeptide research Protein A-gold Technology (PAG method)

Key words: colloidal protein labeling colloidal gold labeling protein a technology

As the second antibody, PAG complex has no species specificity, so it can avoid different species of animals to prepare different specific immunoglobulins. The non covalent binding between protein A and gold particles does not affect the activity of protein A, but also maintains high stability. The PAG complex molecule is the smallest and easy to penetrate tissue. The original solution of PAG complex can be preserved for up to one year at 4 ℃. The principle of PAG staining at the electron microscope level is two-step labeling method, which can be used for staining before and after embedding.

It is mainly different from general colloidal gold immunostaining in that: ① 1% ovalbumin PBS (pH7.4) or 1% ovalbumin-0.05mol/l Tris buffer (pH7.4) is used to seal the nonspecific binding site, instead of using the normal antiserum of sheep or other animals, because the PAg complex can combine with the Ig in the normal serum group, thus giving false positive results; ② when preparing the second antiserum, i.e. the PAg complex, after incubation with the first antiserum and flushing with the PBS, the pH value of the applied PBS or TBS should be changed to that before incubation pH8.2。 Others can refer to the dyeing method after embedding in this section.

Liquid preparation:

1. Formula of colloidal gold diluent:

0.02mo1/l Tris TBS buffer (pH = 8.2) was added with reagent level ovalbumin to 1%. Immunoelectron microscopy should be diluted at 1:20-50, light red is the appropriate diluent, colloidal gold should be used on the same day after dilution, and the unused should be discarded. (immunogold instructions)

1. Cleaning solution:

0.5mol/l, PH7.4 Tris HCl buffer: Tris, 60.57g, 1mol / L HCl about 420m1, adjust the pH value to 7.4, and finally add the double distilled water to 1000m1, which is the reserve solution.

0.05mo1/l Tris buffer normal saline: sodium chloride 8.5-9g; 0.5mo1/l, PH7.4 Tris HCl buffer 100m1; add double distilled water to 1000m1, adjust the pH value to 7.4.

0.02mo1/ltris buffer normal saline: sodium chloride 8.5-9g; o.5mo1/l, PH7.4 Tris HCl buffer 40m1, add double distilled water to l000m1, adjust the pH value to 8.2.

1. Preparation of H2O2: 3% H2O2
2. 8% sodium periodate aqueous solution
3. Sealing fluid:

Pre closure fluid: 1% ovalbumin-0.05mo1/ltris buffer (pH7.4);

After sealing solution and colloidal gold diluent: 1% ovalbumin-0.02mo1/ltris buffer (ph8.2), after sealing, it is prepared for colloidal gold junction cooperation.

Specific operation:

1、 Embedding:

Conventional electron microscopy, the sample only needs glutaraldehyde fixation, does not need osmic acid after fixation. No dye was added to 70% acetone during acetone gradient dehydration. The sample was cured in a 37 degree oven.

2、 Slice:

The ultra-thin section is about 50-70nm thick and is loaded on the nickel mesh with 300 mesh.

Antigen repair:

1. Put it in 1% H2O2 for 10min to 1H (depending on the hardness of the resin and the thickness of the slice), so as to remove osmium acid and improve the penetration of the resin, which is favorable for the entry of antibody. If the slice is very thin or embedded at low temperature, this step can be omitted. During operation, drop 1% H2O2 solution on the wax plate, and float the carrier side of the net on the droplet. It is suggested that 1% potassium periodate (KIO4) should be used instead of H2O2 in the section of central nervous system.

At the same time, 8% sodium periodate water solution was used instead of hydrogen peroxide. The epoxy resin was dissolved at room temperature for 5-25 minutes and acetone. The effect is the best)

1. Wash with double distilled water for 3 times, each time for 10min, the first and second times float on the liquid drop, the third time wash with the syringe containing double distilled water along the nickel mesh surface, the water flow should have appropriate pressure, but should not be too high-strength, use filter paper to absorb the water at the mesh edge. (0.05mo1 / L TBS pH7.4 wash for 5min × 3 times?)

3、 Blocking and immune response:

1. The nickel mesh or gold mesh with ultrathin film was floating on the 1% ovalbumin TBS (7.4) droplet at room temperature for about 1 h, blocking the nonspecific adsorption, absorbing the excess serum and not washing.
2. The carrier network was directly transferred to the first antiserum drop (the dilution of antibody was 1 times lower than that of routine immunohistochemistry), and incubated at room temperature for 2 hours or 4 ℃ for 18-24 hours.
3. 0.05mo1/l TBS pH7.4, wash for 5min × 3 times. 0.02mo1/l TBS ph8.2, wash for 5min × 3 times. 1% ovalbumin 0.02mo1/l TBS (8.2) was incubated at 37 ℃ for 1 h, then the nonspecific adsorption was blocked again, and the excess liquid was absorbed without washing.
4. Dilute PAG stock solution 10-20 times (1:30-1:100, light red is the appropriate diluent), float the net on the drop, and incubate at room temperature for 10 min to 1 h (37 ℃ for 2 h?).
5. 0.02mo1/l TBS ph8.2, wash for 5min × 3 times. 0.05mo1/l TBS pH7.4, wash for 5min × 3 times. Finally, the slices were immersed in 0.05m01/l TBS pH7.4 buffer solution and sent to the electron microscope room for treatment (TBS solution containing 2% glutaraldehyde and 3% paraformaldehyde was fixed for 30 minutes?).

4、 Electron staining and electron microscopy:

1. 5% uranium acetate (prepared with double distilled water) was dyed for 5min, and then washed with double distilled water.
2. Uranium citrate (or lead acetate) was dyed for 5min and washed with double steaming water.
3. H-600 transmission electron microscopy.

Requirements for the success of immunogold test:

The high specificity and affinity of antibodies and the high content of antigens in the tested tissues, the pretreatment and staining of specimens are also very important.

After removal of osmic acid, the polymerization temperature should not be higher than 45 ℃, 37 ℃ 12 hours, 45 ℃ 48 hours can be selected as the polymerization temperature. The whole process of the test shall be kept wet. The buffer should be clean. It is better to use it after fresh preparation or filtration by microporous filter paper.

Before dyeing, all utensils must be clean and special, and washed three times with double steaming water. Washing in dyeing process must be sufficient to reduce background dyeing. In particular, when double immunolabeled sections are stained, they must be washed thoroughly after the first incubation, so as not to affect the reaction results of the second. From: Jia Yunxiang, Zhuo Xiayang, Gu Yundi. Immunoelectron microscopy with double gold labeling. Journal of Jiangxi Medical College, 2003,43 (4): 22-24

The combination of immunohistochemistry and electron microscopy can make use of the advantages of high resolution of electron microscopy to locate specific antigens in the brain at the level of ultramicrostructure. It provides a reliable means for the exploration and research of the etiology, pathogenesis, tissue source, etc. of the disease, and for the improvement of the understanding and diagnosis of the disease.

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