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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
• • Toluidine blue:
• • sodium tetraborate:
• The following Personal Protective Equipment is required for this procedure:
  
  • Lab coat
  • Nitrile gloves (double-layer required; regularly check for holes)
  • Eye gogglesMask
  • 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 – no UA” bag.
• This procedure involves the use of hot plate.  Do not leave the hotplate on unattended.

1.

 Reagents and Supplies

1.1. For Making the Stain Solution

:

• 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:

• Toluidine blue (EMS)
• sodium tetraborate (Fisher)
• purified water (double-distilled, ASTM Type I, or equivalent)
• 20-ml glass vial
• 5-ml syringe
• syringe filter (0.2-um pore size)
• Parafilm (1 × 1 in. piece)
• aluminum foil

1.2. For Staining Semi-thin Sections

• Ultramicrotome and accessories to cut and collect semi-thin sections
• glass microscope slide
• hot plate
• deionized water in a squeeze bottle
• tri-pour beaker to collect liquid waste
• coverslip
• mounting medium (e.g., DPX or Permount)

1.3. Waste Containers

• Liquid waste bottles
  • "Toluidine Blue": By the sink in 3.360J
• Solid waste containers
  • “Solid NaOH” bottle: in “Base” storage container in NHB 3.360E
  • “Solid Waste – No UA” bag: in fume hood in NHB 3.360E

2.  Stain Solution

2.1.  Making Toluidine Blue Solution (estimated work duration: ~30 min)

1. Wear appropriate PPE: lab coat, eye goggles, and nitrile gloves.
2. Place a piece of absorbent sheet on the work surface before starting the procedure.
3. Place a plastic weigh boat on a scale. 
4. Carefully weigh out 0.1 g of Toluidine blue powder into the weigh boat. 
5. Add the Toluidine blue powder into a 20-ml glass vial. Keep the weigh boat.
6. Place a plastic weigh boat on a scale. 
7. Carefully weigh out 0.2 g of sodium tetraborate powder into the weigh boat. 
8. Add the sodium tetraborate powder into the same vial. Keep the weigh boat.
9. Measure out 10 ml of purified water in a graduated cylinder and add the weigh boats to dissolve the remaining powder.  The add the water into the vial.
10. Cover the vial with the cap and shake well.
11. Wrap the vial with a piece of aluminum foil and store at 4C. You can also store the stain solution in a 5-ml syringe with a filter attached – in this case, wrap the tip of the filter to prevent evaporation and wrap the syringe-filter with a piece of aluminum foil before storing at 4C.
12. Dispose of the weigh boat and any contaminated items (incl. absorbent sheet) into the “Solid Waste – No UA” bag. 

2.2. Staining Semi-thin Sections

1. Wear appropriate PPE: lab coat, eye goggles, and nitrile gloves.
2. Place a piece of absorbent sheet on the work surface before starting the procedure.
3. Cut semi-thin

1. sections at 400 or 500 nm.

1. Use a metal loop to collect sections and transfer sections to a drop of

1. purified water on a glass slide.

1. Dry sections on a

1. hot plate.

1. After the sections are completely dried, cover with a drop of filtered Toluidine blue stain solution with the

1. hot plate still on till the edge of the drop

1. starts to dry.

1. Remove from heat and rinse off excess stain gently with

1. water. Collect liquid waste into a tri-pour beaker, then into a "Toluidine blue" waste bottle.
2. Air dry the slide

1. .
2. The stained sections can be observed under a light microscope at this point.
3. Coverslip with regular mounting medium

1. to keep the slide for record.
2. Store the stain solution at 4C.

3.  Clean-up

3.1.  Waste containers:

• Hazardous Liquid Waste: Pour all waste into the proper waste collection bottles available on bench top in NHB 3.360J.
  • “Toluidine blue” (10% nitric acid and first rinse)

3.2.  Glassware and equipment:

• All solid waste items that came in contact with Toluidine blue must be disposed of in the “Solid Waste – No UA” bag.  
• Wipe any small spill with a moistened tissue (e.g., Kimwipe).

Results: