[Histonet] Iron staining (cellular)[Scanned]

[Kemlo Rogerson]

All I can remember is never use an acidic fixative, it removes Fe ions, or
anything acidic before staining. The protocols you suggest appear fine; how
certain are you that the macrophages have 'ingested' iron? Could it be that
cloned cells don't act as they ought and that the iron was never 'ingested'
in the first place? I assume you have put through an Fe2 and Fe3 control
slide to tell you if your procedure was successful?

Kemlo

-----Original Message-----
From: p.j.bergin <@t> qmul.ac.uk [mailto:p.j.bergin <@t> qmul.ac.uk] 
Sent: 15 February 2005 16:29
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Iron staining (cellular)[Scanned]



Hi,

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.

Phil

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

FERROUS IRON - TURNBULL'S BLUE

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.
REAGENTS:
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.

MINERALS AND PIGMENTS
TURNBULL'S FERROUS IRON Page: 2 of 2
Acetic acid: Irritating to respiratory system. Target organ effects on
respiratory system by inhalation. Corrosive.
PROCEDURE:
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.
RESULTS:
Ferrous iron blue
Background pink-red
REFERENCE:
Carson, F, Histotechnology: A Self-Instructional Text, 1st Ed., 1991, pp.
215-16 ASCP Press

Perls Prussian Blue

Principle
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.
Method
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


Notes
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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