Principle and operation steps of ferritin immunoelectron microscopy

(a) Principle

The immunoferrin technology is an antibody labeled with ferritin, and a ferritin antibody acts on the antigen to be detected. By electron microscopy, the location of the ferritin antibody, ie the antigen, was observed.

(2) Materials and reagents

Horse spleen ferritin

2. Ammonium sulfate

3. Cadmium sulfate

4. Metaxylene dlisocyante XC was formulated as a 1% solution with 0.30 Mol/L pH 9.5 borate buffer. Note that the prepared water and container must be specially cleaned and placed at 4 ° C for several days after preparation, which is subject to no precipitation. If a polymer with precipitated XC is formed, it should be reconstituted.

5.0.30Mol/L pH 9.5 Boric Acid Buffer

6.0.1Mol/L ammonium sulfate solution

7.0.05Mol/L pH 7.4 PBS solution

(3) Operation method

Ferritin extraction

(1) Prepare 2% ammonium sulfate solution and adjust the pH with 1 Mol/L NaOH or HCl, which is exactly 5.85. 1 g of ferritin was dissolved in 100 ml of 2% ammonium sulfate solution.

(2) Add 20% cadmium sulfate to a final concentration of 5%, mix and mix at 4 °C overnight.

(3) Centrifuge (4 ° C) for 1 h at 1 500 g, and remove the supernatant. Still add 2% ammonium sulfate to 100ml, mix and centrifuge to remove impure sediment.

(4) Re-add 20% cadmium sulfate to the supernatant, repeat step (2), centrifuge, and remove the supernatant.

(5) Check the sediment and check it under the Microscope. It should have a typical yellow-brown crystal, the crystal is hexagonal, and the double-four-point structure. If the crystal is not typical, the above steps should be repeated.

(6) Dissolve in a small amount of distilled water, add 50% saturated ammonium sulfate solution, precipitate it, centrifuge, and remove the supernatant.

(7) Repeat step (6) once.

(8) Dissolved in a small amount of distilled water, dialyzed for 24 h in normal water, and dialyzed against 0.05 Mol/L pH 7.5 PBS for 24 h.

(9) Centrifuge at 100 000 r/min for 2 h, remove the upper colorless supernatant (about 3/4 total), and set at 4 ° C overnight.

(10) Filter with a microporous membrane (pore size 0.45μ) to make the ferritin content from 65mg/ml to 75mg/ml, dispense, and store at 4°C without lyophilization to avoid damage to the ferritin structure.

2. Ferritin-antibody cross-linking method

(1) The ferritin was diluted to 20 mg/ml to 25 mg/ml in a 0.3 Mol/L pH 9.5 borate buffer.

(2) Add XC solution at a ratio of 1..1000 (W/W), stir at room temperature for 45 min, and centrifuge to remove.

(3) The purified lgG was formulated into 5 mg/ml in a 0.3 Mol/L pH 9.5 borate buffer, and IgG and ferritin-XC solution were added at a ratio of 1..4 (V/V), and stirred at 4 ° C for 48 hours.

(4) Dialysis was carried out overnight with 0.1 Mol/L ammonium carbonate solution to remove excess isocyanate, and then dialyzed against 0.05 Mol/l pH 7.5 PBS to restore the pH to physiological level.

(5) Centrifugation was performed at 1.00 × 105 g for 5 h, the supernatant was removed, the precipitate was suspended in 0.05 Mol/L PBS, and centrifuged again to remove unbound IgG.

(6) The specificity, immunological activity, and labeling effect of the bound antibody are determined by serological and immunological methods. If the procedure is strict, the results are satisfactory.

3. Ferritin-antibody conjugate treatment specimen

(1) The specimen was fixed in 5% formalin pH 7.2 (4 ° C) PBS for 40 min to 60 min.

(2) Wash with cold PBS solution and centrifuge.

(3) If it is a tissue block, cut it into smaller pieces under a dissecting microscope, put it into a test tube, add ferritin-antibody conjugate and let it stand at room temperature for 20 min, occasionally oscillate.

(4) Wash three times with cold PBS and centrifuge.

(5) The pellet was fixed with 2.5% glutaraldehyde for 20 min and washed with PBS.

(6) Fix it with citric acid and dehydrate it.

It is also possible to perform ultrathin sectioning and then staining for ferritin-antibody conjugates. The operation is as follows:

(1) The cultured cells were fixed in 1% formalin PBS (4 ° C).

(2) Wash the PBS solution by centrifugation.

(3) Dissolve in 0.5ml, 30% bovine serum albumin PBS solution in a plastic dialysis membrane bag, and then place the bag on the water absorbing agent powder. When the bovine serum albumin is gelatinized, the dialysis bag is moved to 2% pentane. The dialdehyde PBS solution (pH 7.5) was fixed for 3 hours.

(4) Take out, cut into small pieces, and wash with PBS solution.

(5) Dry in a desiccator with silica gel.

(6) Embedding, sectioning, and collecting slices on the water on a film coated with 4% bovine serum albumin PBS (the treatment of bovine serum albumin is to reduce the non-specific adsorption of ferritin conjugates on the carrier) .

(7) Drop a drop of ferritin-antibody conjugate on the loading grid.

(8) After 5 minutes, float the net on the PBS surface and the specimen face down to remove excess conjugate.

(9) After drying, drop a drop of uranyl acetate or lead hydroxide to counterstain.

(10) Washing, drying, and electron microscopic observation.

(4) Result determination

On the premise that the known control sample is established, any black iron particle that appears to be black indicates the presence of the antigen, and the positive (+) is judged as negative (-).

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