eTraining Introduction

Specimen Preparation

Leica Ultracut UCT

Histology

JEOL JSM-6400 SEM

Hitachi S-4700 FE-SEM

Hitachi FB-2000A FIB

Microanalysis

Veeco Dim 3000 AFM

Fluorescence Microscopes

Support

Procedure

Solution Preparation

Prepare all needed chemicals ahead of time, except the ethanol grades. Store in the refrigerator if not used right away. Seal all beakers tightly with Parafilm. All solutions, except the physiological saline, should be made under a fume hood. It is essential that all glassware and tools coming into contact with the chemicals should be clean. Wash everything with a soap, such as Micro-90, and rinse with distilled water at least three times.

Physiological Saline

  1. In a 1 L jar with a lid, add 300 mL of dH2O
  2. Add the following solids to a jar:

    8.0 g NaCl
    0.2 g KCl
    0.2 g CaCl2
    0.15 g MgCl2
    1.0 g NaHCO3
    0.04 g NaH2PO4
    1.0 g Sucrose

  3. Place the jar on a stir plate and add a CLEAN stir bar to the jar.
  4. Dissolve the solids and bring the final volume to 1 L.
  5. Wrap Parafilm around the lid to improve the seal and place the jar in refrigerator.

Fixative: 2% glutaraldehyde in 0.1 M Na-cacodylate buffer with 0.1 M sucrose

VIEW THE SAMPLE CALCULATIONS

The unit M is molarity, or mol/L.

  1. Add 4.28 g Na-Caco to 100 mL of dH2O. Check pH with meter.
  2. Add 0.2 M HCl by pipette until pH reaches ~7.0.
  3. Add distilled water to a final volume of 200 mL to create a 0.1 M solution.
  4. SPLIT SOLUTION IN HALF! The second 100 mL of solution will be used to make the post-fixative.
  5. To 76.5 mL of one of the two portions add 23.5 mL of 8% glutaraldehyde to make a 2% glutaradehyde solution.
  6. Add 3.42 g sucrose and stir well until dissolved.

Treatment: 1% tannic acid in 0.15 M Na-cacodylate buffer

VIEW THE SAMPLE CALCULATIONS

  1. To make a 1% solution, just add 1 g of Tannic acid to 100 mL dH2O. The ratio is not exact, but it is very close.
  2. Add 3.21 g of Na-Cacodylate powder to the 100 mL solution.

Post-fixative: 1% osmium tetroxide in 0.1 M Na-cacodylate buffer

VIEW THE SAMPLE CALCULATIONS

  1. To the other previously made (for the fixative solution) 100 mL of 0.1M Na-Caco buffer: Take out 12.5 mL of it and put in separate beaker.
  2. To that 12.5 mL add 12.5 mL of 2% OsO4.

There should be an excess of the buffer. It is important to minimize the amount of osmium tetroxide used because it is a very toxic chemical. Therefore, very little of the prepared buffer will actually be used.

Ethanol series

VIEW THE SAMPLE CALCULATIONS

Make a graded series of ethanol (EtOH) in volumes of 40 mL each using 95% (190 proof) ethanol. Seal each beaker securely with Parafilm once the solution is made. Use concentrations of 30%, 40%, 50%, 70%, 80%, and 90%.

The final wash will be 100% anhydrous ethanol. Once you open the anhydrous ethanol, it cannot again be considered 100% ethanol.

To make each solution, add the specified amount of ethanol to an empty beaker and the correct difference of dH2O to make 40 mL of total solution.

40% = 16.8 ml of 95% EtOH
50% = 21.05 ml of 95% EtOH
70% = 29.5 ml of 95% EtOH
80% = 33.7 ml of 95% EtOH
90% = 37.9 ml of 95% EtOH

Be sure to tightly seal each wash with Parafilm.

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