Experiments on the electron microscopic display of cell membrane glycans

Summary

Cell membrane glycans electron microscopy display experiments can be used to: observe glycoproteins and glycolipids on cell membranes. The more commonly used display technique is lectin cytochemistry, in addition to ruthenium red staining.

Operation method

ruthenium red staining

Principle

The ruthenium red staining method is based on the principle that ruthenium red can combine with carboxyl groups (acidic groups) by electrostatic binding, which shows high electron density staining under the electron microscope.

Materials and Instruments

Tissue samples
Ruthenium red stock solution Glutaraldehyde Sodium dimethylarsinate buffer Osmium acid
Pipette gun

Move

1. Preparation of ruthenium red stock solution

Ruthenium red was dissolved in double-distilled water at a concentration of 10 mg/ml at 60℃, shaken for 10 min, centrifuged at 3000 r/min for about 15 min, and the supernatant was taken and stored under refrigeration.

2. pre-fixation solution 3% glutaraldehyde 2.5 ml, 0.2 mol/L sodium dimethylarsinate buffer (pH 7.3) 2.5 ml, ruthenium red stock solution 2.5 ml, mixed in order.

3. Post-fixation solution 2% osmium 2.5 ml, 0.2 mol/L sodium dimethylarsinate buffer (pH 7.3) 2.5 ml, ruthenium red stock solution 2.5 ml are mixed sequentially.

4. The specimens were cut into small pieces (1 mm³ ) and placed in the pre-fixation solution for 1 h at room temperature.

5. 0.1 mol/L sodium dimethylarsinate buffer was rinsed 3 times for 3 min each. 6.

6. 1 % osmium post-fixation solution was fixed at room temperature for 2 h. 7.

7. Rinsing, dehydration, and embedding were performed as usual.

Caveat

Cytoplasmic membranes stained with heavy metal dyes such as ruthenium red show structures about 10-20 nm thick with poorly defined borders under the electron microscope.

Common Problems

Stains mycelium and spores in host plant tissues in plant pest and disease tissue cut-pin staining.

Sourced from Experimental Techniques and Applications of Immunohistochemistry

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