Deep-stain embedded 方法

Materials:
1.Ammonium molybdate
2.Trehalose

Negative stain solution:
1.16% (w/v) Ammonium molybdate or
2.4% (w/v) Ammonium molybdate with 1% (w/v) Trehalose at pH=7.0

Solution preparation (always prepare in fresh):
1.Take 0.16 mg ammonium molybdate in an eppendorf tube
2.Add 0.8 mL distilled water into the eppendorf to dissolve ammonium molybdate
3.Add few drops of 10N NaOH and use pH paper to adjust the proper pH
4.Add distilled water to a final volume of 1 mL
5.Use 1 mL syringe and 0.2 um syringe filter to filter the negative stain solution


For 4% (w/v) Ammonium molybdate w/ 1% (w/v) Trehalose:
6.Take 0.16 mg ammonium molybdate and 0.01 mg Trehalose in an eppendorf tube
7.Follow the same preparation steps described above.

Stain protocol:
1.Put ~50 μL drop of stain solution on a parafilm
2.Apply 3 μL sample onto a glow-discharged holey carbon grid (continuous carbon coated grid also works fine) and wait for 25 seconds.
3.Use filter paper to blot the sample solution from the edge of grid.
4.Immediately place the grid on the top of the stain solution with the sample side facing to the stain
5.Wait for 10 seconds and blot the stain with filter paper
6.At this point, one should be able to see a disappearance process of the stain on the grid surface
7.If the surface remains wet, shake the grid to let it dry faster
8.Immediately inspect the specimen under the electron microscopy