Re: [Histonet] Iron staining (cellular)[Scanned]

From:Geoff McAuliffe

    Kelmo has a good point here, cultured macrophages may not have iron 
in them. Not every macrophage in the spleen has iron deposits, some are 
newly formed from monocytes or cell division. And, cells in culture do 
what they want which is not always what we hope for. For control, I 
suggest sections of spleen, not a few dabs of cells on a slide.
    Prussian and Turnbull blue are easy to do so it is unlikely that the 
problem lies with you. Do be sure to mix the HCl +  
ferrocyanide/ferricyanide immediately before use, not just fresh that day.

Geoff

Kemlo Rogerson wrote:

>You must have a control slide or you will never get to the bottom of it. Why
>should the macrophages have iron in them? When I cultured blood cells for my
>MSc I learnt very quickly that what you got may bear no resemblance to what
>you started with; cells that posses certain characteristics in vivo may not
>in vitro. I wonder if you can't stain it as it's not there; haemorrhagic
>placenta or omentum, I'm told, is a good control (Dave Rushworth; verbal).
>
>
>That fellow J.A. Kiernan has a good section in his book 'Histological and
>Histochemical Methods' Theory & Practice, 3rd Edition, Published by Arnold
>1999.
>
>Kemlo Rogerson
>Cellular Pathology Manager
>East Lancashire Hospitals NHS Trust
>DD. 01254-294162
>Mobile 0774-9754194
> 
>
>-----Original Message-----
>From: p.j.bergin@qmul.ac.uk [mailto:p.j.bergin@qmul.ac.uk] 
>Sent: 16 February 2005 11:25
>To: Kemlo Rogerson
>Cc: histonet@lists.utsouthwestern.edu
>Subject: RE: [Histonet] Iron staining (cellular)[Scanned]
>
>I thought so too (about the adic fixing).  So far we have fixed in
>methenol/acetone, NBF, and tried with just air drying.  The cells themselves
>are just cultured macrophages (primary) and should have iron in them. 
>Unfortunaetlywe have no control, but have been using murine spleen cell
>smears
>for the positive and to try get the assay working.  Still no luck though. 
>Although if you have a better suggestion for a control slide then that'd be
>great.
>
>Cheers,
>
>Phil
>
>Quoting 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@qmul.ac.uk [mailto:p.j.bergin@qmul.ac.uk]
>>Sent: 15 February 2005 16:29
>>To: histonet@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 4.
>>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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>>
>>    
>>
>
>
>
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>  
>

-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff@umdnj.edu
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