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.
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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.
ReagentsReagents 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:
1. Cut semithin- 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)
- Wear appropriate PPE: lab coat, eye goggles, and nitrile gloves.
- Place a piece of absorbent sheet on the work surface before starting the procedure.
- Place a plastic weigh boat on a scale.
- Carefully weigh out 0.1 g of Toluidine blue powder into the weigh boat.
- Add the Toluidine blue powder into a 20-ml glass vial. Keep the weigh boat.
- Place a plastic weigh boat on a scale.
- Carefully weigh out 0.2 g of sodium tetraborate powder into the weigh boat.
- Add the sodium tetraborate powder into the same vial. Keep the weigh boat.
- 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.
- Cover the vial with the cap and shake well.
- 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.
- 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
- Wear appropriate PPE: lab coat, eye goggles, and nitrile gloves.
- Place a piece of absorbent sheet on the work surface before starting the procedure.
- Cut semi-thin sections at 400 or 500 nm.
- Use a metal loop to collect sections and transfer sections to a drop of
- purified water on a glass slide.
- Dry sections on a
- hot plate.
- After the sections are completely dried, cover with a drop of filtered Toluidine blue stain solution with the
- hot plate still on till the edge of the drop
- starts to dry.
- Remove from heat and rinse off excess stain gently with
- water. Collect liquid waste into a tri-pour beaker, then into a "Toluidine blue" waste bottle.
- Air dry the slide
- .
- The stained sections can be observed under a light microscope at this point.
- Coverslip with regular mounting medium
Results:
Cells and nuclei stained blue- to keep the slide for record.
- 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).