Johnson's Method For Iron (Microincineration)
EMS Catalog #26603
10% Buffered Neutral Formalin
Paraffin at 6 microns
- 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.
- 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.
- After the slide has cooled, the completely incinerated specimen is examined under strong oblique lighting with dark field illumination, with or without coverslip.
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
- 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.
- 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.
- Air dry or dry in a 60°C oven.
- Mount with Permount.
C. Direct observation of Prussian Blue Reaction on the Ash
- Create a well paraffin around the specimen.
- 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)
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.