Re: Staining of collagen - on frozen sections

From:=?iso-8859-1?q?Steve=20Machin=20UK?= <>

Here is a method written by M.L. used in my lab.

Use  The demonstration of collagen proliferation.

1. Air dry sections for at least 10 minutes or longer.
2. Place cryostat sections into 10% buffered formalin (COSHH I) at room
temperature (for 10 mins or until convenient to stain).
3. Wash well in tap water.
4. Stain nuclei with Weigert's iron haematoxylin for 5 minutes.
5. Rinse in water
6. Differentiate in 1% acid alcohol for 2 seconds.
7. Wash in tap water and rinse in distilled water.
8. Stain in Van Gieson solution for 5 minutes.
9. Drain slide and wipe off excess stain but do not wash in water.
10. Rapidly dehydrate in 99% alcohol only 
11. Clear in xylene and mount

Weigert's iron haematoxylin
Add equal parts of solutions A and B immediately before use.  This mixture will
keep for a few hours only.  It should be a violet black colour and must be
discarded if brown.
(Both solutions are to be found in the cupboard under the window bench in the
main lab).

Weigert's Solution A 

Haematoxylin  	10 g
Absolute alcohol  (COSHH H I) HF	1000 ml

Solution B 
30% aqueous ferric chloride (anhyd.) 	40 ml
Concentrated hydrochloric acid  	10 ml
Distilled water	950 ml

Van Gieson solution

Saturated aqueous picric acid 	95 ml
1% acid fuchsin (C.I. 42685)  in distilled water	5 ml

The mixture keeps well.

Nuclear staining is readily discoloured by picric acid in the Van Gieson stain.
 Therefore, only very brief  differentiation in acid alcohol following iron
haematoxylin staining is necessary.
Red staining of tissue is removed if sections are washed in water after Van
Gieson's stain.
Yellow staining is removed during dehydration therefore this procedure must be

Best Wishes
Steve Machin UK

--- wrote: > Hi Histonetters
> I posted this question last week, but I got no response, so I will try
> again in hope of some.
> Does anyone have a protocol for Van Gieson staining on frozen sections.
> Alternatives to this staining method for collagen are also welcome as long
> as they work on frozen sections. I need it for a 3D in vitro skin model
> growing in a matrix that cannot be formalin fixed and paraffin embedded.
> Dorte

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