by Weiling Yin
Toluidine blue is used for staining of semithin sections. The stained section is used as a guideline to determine the area of interest and further trimming of embedded tissue block.
0. Safety Precautions:
- You must complete required lab safety training before starting this procedure.
- If this is your first time doing this procedure, ask to be trained by an experienced lab member. If you have not done this in a while, you should ask for a refresher.
- Before starting, even if you have done this procedure before,
- read this protocol entirely
- review relevant Safety Data Sheets and Harris Lab SOP (also see below)
- ensure you have all reagents and supplies listed below
- ensure all equipment is in good working order
- have all waste containers ready (also see Clean-up)
- plan your schedule well so that you wouldn’t have to rush
- Review SDS and Harris Lab SOP for the following hazardous chemicals used in this procedure:
- Nitric acid: strong oxidizer; corrosive; causes severe skin burns and eye damage; irritant (respiratory)
- Lead nitrate: oxidizer; toxic (ingestion, inhalation); carcinogen; reproductive toxin; causes serious eye damage; environmental toxin
- Sodium hydroxide: corrosive; causes severe skin burns and eye damage; irritant (respiratory)
- Uranyl acetate: fatal (inhalation, ingestion); flammable
- The following Personal Protective Equipment is required for this procedure:
- Lab coat
- Nitrile gloves (double-layer required; regularly check for holes)
- Eye goggles
- Mask
- OPTIONAL: plastic apron, shoulder-length gloves, lead glass shield
- Place a piece of absorbent sheet on the work surface before starting the procedure. When done, discard in the “Solid Waste – UA” bag.
- This procedure involves the use of hot plate. Do not leave the hotplate on unattended.
Reagents:
- 1% Toluidine blue and 2% Borate in distilled water
- Toluidine blue: 0.1 g
- Sodium borate (Borax): 0.2 g
- Distilled water: 10 ml
- Store the filtered solution in light protected bottle in 4C as stock stain solution.
- Use a 1 ml syringe for staining procedure (Remember to attach a filter on syringe when use).
Procedure:
1. Cut semithin sections at 400 or 500 nm.
2. Use a metal loop to collect sections and transfer sections to a drop of distilled water on a glass slide.
3. Dry sections on a heat plate.
4. After the sections are completely dried, cover with a drop of Toluidine blue with the heat source still on till the edge of the drop start to dry.
5. Rinse off excess stain gently with distilled water
6. Air dry the slide
(7. Coverslip with regular mounting medium if slide need to be kept for record.)
Results:
Cells and nuclei stained blue