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Electron Microscopy Sciences

Technical Data Sheets

Johnson's Method For Iron (Microincineration)

EMS Catalog #26603

Fixation

10% Buffered Neutral Formalin

Sections

Paraffin at 6 microns

Staining

A. Microincineration

  1. Place unstained paraffin section uncovered on a sheet of asbestos and heat cautiously, using the outer cone from the flame of a Fisher or Meker burner. Keep the flame in motion and heat the slide as uniformly as possible.
  2. Continue the heating until the specimen, which turns brown then white, has almost completely disappeared. Immediately cover the slide with an inverted pan, (e.g. a pie pan) and allow the slide to cool gradually.to rapid cooling may cause the slide to break.
  3. After the slide has cooled, the completely incinerated specimen is examined under strong oblique lighting with dark field illumination, with or without coverslip.

    Results
    Iron oxide imparts a bright yellow-orange to dark red color to the ash. Specimen must be completely incinerated as carbon particles can also give a yellowish to red color.

B. Staining of Incinerated Specimen

  1. Dip incinerated specimen in a thin celloidin solution (approx. 0.015% in alcohol: ether, 1:1). Air dry slides for a few minutes and then quickly hydrate to distilled water.
  2. Stain in working hydrochloric acid-potassium ferracyanide solution* for 15 minutes. Rinse in several changes of distilled water. *Prepare working stain by mixing equal parts of 5% hydrochloric acid and 5% potassium ferracyanide just before use.
  3. Air dry or dry in a 60°C oven.
  4. Mount with Permount.

C. Direct observation of Prussian Blue Reaction on the Ash

  1. Create a well paraffin around the specimen.
  2. Pour working Hydrochloric Acid-Potassium Ferracyanide Solution (for preparation, sheet step B-2) into the paraffin well and observe under the microscope.

Staining Results (B & C)

Iron Oxide Blue

 

References

Fenton, R.H., Johnson, F.B., and Zimmerman, L.E., J. Histo-chem. Cytochem ., 12:153:155, 1964.

L una, L.G., (ed.), Manual of Histologic Staining Methods of the AFIP , 3 rd edition, McGraw-Hill, NY., p. 181., c 1968.

Online Ordering

Potassium Ferrocyanide Sol 5% and Hydrochloric Acid 5% for Johnson's Method for Iron (Microincineration) are available online from the EMS Catalog. For ordering or product information, click here.