Materials and Reagents

Control: A known positive control tissue (such as spleen)
Formalin
EtOH
Hydrochloric acid
Aluminum sulfate
Histoclear reagent
Mounting solution
Fixative (see Recipes)
5 % potassium Ferrocyanide (see Recipes)
5% hydrochloric acid (see Recipes)
Nuclear-fast red (Kernechtrot) (see Recipes)
Working solution (see Recipes)

Equipment

Microwave oven
Acid-cleaned glassware
Non-metallic forceps.
Gloves, goggles and lab coat
Fume hood

Procedure

Safety: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Potassium ferrocyanide; Low toxicity as long as it is not heated, it will release cyanide gas.
Hydrochloric acid; target organ effects on reproductive system and fetal tissue. Irritant to skin eyes and respiratory system.

Deparaffinize tissue slide and hydrate to distilled water (use Histoclear reagent 2x, 100% EtOH 2x, 95% EtOH 1x, 90% EtOH 1x, 80% EtOH 1x, 70% EtOH 1x, ddH₂O 1x. leave 3-5 min in each solution).
Immediately transfer slides into working solution (avoid drying the tissue) and either use the microwave method (30 sec, then allow slides to stand in solution for 5 min in fume hood) or use the conventional method (room temperature for 30 min).
Rinse in distilled water.
Nuclear-fast red (incubate for 5 min at room temperature).
Wash in tap water (several washes).
Dehydrate, clear (reverse the order of step 1 above).
Add mounting solution and coverslip.
Results:
Iron (hemosiderin):
Blue
Nuclei:
Red
Background:
Pink
(Slide shows mouse spleen at 400x)

Representative data

Figure 1. Typical results from this experiment

Recipes

Fixative
10% formalin
5% potassium ferrocyanide
Potassium ferrocyanide 25.0 mg
Distilled water 500 ml
Mix well, pour into an acid-cleaned brown bottle.
Stable for 6 months.
Caution: Low toxicity if not heated.
5% hydrochloric acid
Hydrochloric acid, conc. 25.0 ml
Distilled water 475.0 ml
Mix well, pour into brown bottle.
Stable for 6 months.
Caution: Corrosive, avoid contact and inhalation.
Nuclear-fast red (Kernechtrot)
Aluminum sulfate 25.0 mg
Distilled water 500 ml
Nuclear-fast red 0.5 mg
Dissolve the aluminum sulfate in distilled water, then the nuclearfast red, using heat. Cool, filter, and add a few grains of thymol as a preservative.
Label with date. Stable for 1 year.
Caution: Irritant, avoid contact and inhalation.
Working solution
5% potassium ferrocyanide 25.0 ml
5% hydrochloric acid 25.0 ml
Make fresh, discard after use.
Caution: Avoid contact and inhalation.

Acknowledgments

This protocol was adapted/modified from original versions as described in Luna (1968).

References

Sheehan, D., Hrapchak, B. (1980). Theory and practice of histotechnology. 2nd ed.; Battelle Press: Ohio, p. 217-218.
Luna, L. (1968). Manual of histologic staining methods of the AFIP. 3rd ed.; McGraw-Hill: NY. P. 183.
Crookham, J., Dapson, R. (1991). Hazardous chemicals in the histopathology laboratory. 2nd ed.; Anatech.