Description: This method is used for staining of TEM thick sections. The stained sections can be used as guideline to determine the area of interest and further trimming of Embed blocks. Therefore precise ultra-thin sections can be cut and mounted on TEM grids. This stain can also be used for staining of plastic sections of small tissue samples such as peripheral nerve, sciatic nerve, etc. for general morphological analysis.

Fixation: 4% formaldehyde and 1% glutaraldehyde in phosphate buffer.Reagents:

1% Toluidine Blue and 2% Borate in Distilled Water

   Toluidine Blue O ---------------------------- 1 g

   Sodium Borate (Borax) --------------------- 2 g

   Distilled Water ------------------------------ 100 ml

Dissolve the sodium borate in the water, then add the toluidine blue powder and stir until dissolved. Filter the stain solution (use syringe filter) before use (Note: The borax makes the stain alkaline so it will help penetrating to the epoxy sections).Procedure:

1. Cut thick (semithin) sections at 0.5 um or 1.0 um.

2. Use a metal loop to collect thick sections, and transfer sections to a drop of distilled water on a glass slide.

3. Dry sections down on a glass slide by placing the slide on a slide warmer or 40 wt lamp.

4. After the sections are completely dried, cover with a few drops of staining solution (with the heat source still on) for 1-2 minutes depending on the darkness of staining you would like to achieve.

5. Rinse off excess stain gently with distilled water.

6. Air dry the slide.

7. Coverslip with regular mounting medium.

Results:

Cells and nuclei stained blue.

References:

1. Mercer, E.H., J. Royal Microscopy Society 81:179 (1963).

2. Burns, W.A., "Thick Sections: Technique and Applications", Diagnostic Electron Microscopy, Ch. 4, B.F. Trump and R.J. Jones, eds., John Wiley & Sons, New York, 1978