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Electron Microscopy Facility
Subsidiary of the Research and Graduate Studies Office of the AVP

Staining for Semi-Thin (LM) Sections

June 08, 2023 04:23 PM

Stain Preparation for Semi-Thin Sections:

  1. Obtain the previously used erlenmeyer flask with magnetic stir bar (don't get a new/clean flask)
  2. Wash out the excess stain from the flask
  3. Dry the outside of the flask
  4. Add 30 ml deionized (D.I.) water to the flask
  5. Add 0.3 gm Toluidine Blue O to the flask
  6. Add 0.3 gm Azure II to the flask
  7. Add 0.3 gm Sodium Borate to the flask
  8. Heat the flask while stirring on a hot plate for 5 minutes until contents are fully dissolved and stain is approximately 60 to 70 degrees C.

NOTES: 1) This stain will stain the glass and stir-bar so please reuse the flask and stir-bar that is already stained. 2) Do not allow the stain to boil when heating as this will cause a mess that cannot be fully cleaned. 3) The general recipe for this stain is 1% Toluidine Blue O: 1% Azure II: 1% Sodium Borate in D.I. water (w/v).

Staining Technique:

  1. Warm prepared slides on a hot plate to approximately 60 to 70 degrees C.
  2. Flood the area on the slide that contains the semi-thin sections with the stain.
  3. Let sit for 1 minute. Do not allow stain to boil or to dry out.
  4. Rinse staining solution off of each slide using a stream of D.I. water. Do not let the stream of water hit the sections, rather make the water flow across the sections from a point above the sections on the slide (See Mike if this doesn't make sense).
  5. Dry the underside of the slide (not the side with the sections) with a kimwipe then put slides back on the hot plate to dry completely.

Applying Coverslips to the Slides:

  1. Place a slide on an absorbent surface i.e. a stack of kimwipes, paper towles, etc.
  2. Using an applicator stick, put a drop of Permount on the slide over the sections.
  3. Carefully lower one edge of a coverslip to the surface of the slide, then using a clean applicator stick carefully lower the other edge of the coverslip onto the slide.
  4. Using a clean applicator stick, press down on the coverslip to help distribute the Permount and to remove any air bubbles.
  5. Carefully blot the long edge of the slide closest to you on the absorbent surface to remove excess Permount then put slide in an out of the way place to dry overnight.
  6. Put glass weights on the coverslip to help keep sections from wrinkling.