[Histonet] Iron staining (cellular)

p.j.bergin <@t> qmul.ac.uk p.j.bergin <@t> qmul.ac.uk
Tue Feb 15 10:28:39 CST 2005


I was hoping someone could give me advice.  I have looked through the archives
and have tried a few things already.  I am interested in staining cell preps to
localize iron.  I have tried using mouse spleen cells as a trial, but am mainly
interested in human macrophages and monocytes that have been grown on glass
slides.  I have tried fixing the preps with a methanol/acetone mix and with 4%
formalin.  I have been trying two different methods; Perls Prussian blue from
the stainsfile archive and Turnbull's Blue (for ferric and ferrous iron
respectively).  Both have been unsuccessful with no staining detected (and the
cells are there).

Does anyone have a method for these stains that works on cells? do they need to
be fixed a specific way? Is there any advice anyone would care to share?

Thanks in advance for any help.


P.S. The staining protocols I have been using follow.....


PURPOSE: To detect ferrous (Fe2+) iron in tissues.
PRINCIPLE: Tissue sections are treated with an acidic solution of potassium
ferricyanide, any ferrous iron present will react to form an insoluble bright
blue pigment called Turnbull’s blue (ferrous ferricyanide).
CONTROL: Routine iron control, which includes an negative.
TECHNIQUE: Cut paraffin section 4µ.
EQUIPMENT: Acid clean glassware, non-metallic forceps.
0.006N Hydrochloric Acid 1% Acetic Acid
Hydrochloric acid 2.5 ml Acetic acid, glacial 1.0 ml Distilled water 497.5 ml
Distilled water 100.0 ml Mix well. Solution is stable for 1 Mix well. Solution
is stable for 1 year. year.
CAUTION: Corrosive acid. CAUTION: Avoid contact and inhalation.
Potassium Ferricyanide Nuclear-Fast Red:
Staining Solution: See Retic
Potassium ferricyanide 0.4 gm
Hydrochloric acid, 0.006N 40.0 ml
Prepare fresh, just before use.
CAUTION: Avoid contact and inhalation.
SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation.
Hydrochloric acid; target organ effects on reproductive system and fetal tissue.
Irritant to skin eyes and respiratory system.
Potassium ferricyanide; Low toxicity as long as it is not heated, it will
release cyanide gas.

Acetic acid: Irritating to respiratory system. Target organ effects on
respiratory system by inhalation. Corrosive.
1. Deparaffinize and hydrate to distilled water.
2. Place slides in Potassium ferricyanide staining solution for 1 hour.
3. Wash slides in 1% acetic acid.
4. Counterstain slides in nuclear-fast red for 5 minutes.
5. Rinse well in distilled water.
6. Dehydrate, clear and coverslip.
Ferrous iron blue
Background pink-red
Carson, F, Histotechnology: A Self-Instructional Text, 1st Ed., 1991, pp.
215-16 ASCP Press

Perls Prussian Blue

The basis for the method is the release of ferric iron from hemosiderin by acid
treatment, forming ferric chloride. The ferric iron reacts with potassium
ferrocyanide to form ferric ferrocyanide. This is an insoluble, blue compound
known as Prussian blue. The intensity of the colour gives some indication as to
amount, but it is qualitative only.

4FeCl3 + 3K4Fe(CN)6 = Fe4[Fe(CN)6]3 + 12KCl

  Stock solution A
 Hydrochloric acid 2 ml
 Distilled water 98 ml

  Stock solution B
 Potassium ferrocyanide 2 g
 Distilled water 100 ml

  Working solution C
 Solution A 1 part
 Solution B 1 part

  Counterstain solution D
 Neutral red counterstain

Fixation & processing
Avoid iron containing materials and jars while fixing as these may contaminate
the tissue. Acid containing fixatives may remove some of the iron deposits.
Otherwise most methods are satisfactory.
Bring sections to distilled water with xylene and ethanol.
Place into working solution C for 15 minutes.
Rinse with distilled water, then tap water.
Stain with counterstain solution D for one minute Rinse well with tap water.
Dehydrate with ethanol.
Clear with xylene

Expected Results
Nuclei - red

The working solution C must be made immediately before use.
Avoid washing with tap water before treating with solution C, as rust in the
water or tap fixtures could cause false positive staining.
Wash well at step 3, as traces of iron will form a granular red deposit with
neutral red.
Iron ores can be demonstrated, but the acid concentration in solution A may need
to be increased to 10% or more.

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