[Histonet] Collagen Staining

[Paul Bradbury]

Hi Jo-Ann,

I think you are working in the right direction, you just need to be a 
bit more aggressive with the heteropolyacids to avoid any non-collagen 
staining. A number of years ago, I did quite a lot of work on the 
effects of various heteropolyacids on trichrome and fibrin stains.

Phosphomolybdic acid is the most commonly used heteropolyacid, but there 
are several others with similar properties. Most labs have some 
phosphotungstic acid on hand. Some of the others I worked with were 
borotungstic acid and silicotungstic acid.

I would be tempted to first try pre-treating the sections with 5% 
phosphotungstic acid for 5-10 minutes and incorporating 5% PTA in the 
stain solution as well. No washing between the two solutions.

Can you tweak the imaging program or the illumination to over-ride very 
pale reactions?

I am keeping my fingers crossed,

Paul

>Hi Paul,
>
>I guess I should have been a little more descriptive.  That is exactly what I did.  The problem is the background retains enough of a blue tinge to interfere with the imaging program they are using.
>
>Jo-Ann Bader
>
>
>
>-----Original Message-----
>From: Paul Bradbury [mailto:histology.bc <@t> shaw.ca]
>Sent: Mon 3/5/2007 10:05 AM
>To: Jo-Ann Bader, Ms.; HistoNet Server
>Subject: Re: [Histonet] Collagen Staining
> 
>Trying to stain collagen by using just the aniline blue solution from a 
>trichrome method will not work. An acidified solution of aniline blue 
>will non-selectively stain all acidophilic tissues (red cells, muscle, 
>cytoplasm, collagen, etc.).
>The reason that the trichrome methods distinguish collagen from other 
>tissue structures is that red cells and muscle have been pretreated with 
>phosphomolybdic acid prior to staining in aniline blue. The presence of 
>the aniline blue in those tissues inhibits (for a period of  time) the 
>attachment of the blue dye. The principles of the trichrome techniques 
>depend on the molecular size of the dye molecules and the relative 
>porosity of the tissue proteins that are to be stained.
>
>To selectively stain collagen without any other background staining, I 
>would suggest trying the following.
>
>Bring the sections down to water as usual.
>Treat the sections in 1% aqeous phosphomolybdic acid for 5 minutes
>Drain off the acid. Do not wash as this will extract the PMA from the 
>tissues.
>Stain in acidified aniline blue (or any one of the blue or green stain 
>solutions used in a trichrome method) for 3 minutes.
>Rinse very briefly in distilled water. Just a few seconds, no longer.
>Dehydrate quickly through graded alcohols.
>Clear in xylene and mount.
>
>What I believe will happen is that the PMA will block entry of the dye 
>to all tissue structures except collagen, thus giving selective 
>coloration of the collagen fibres.
>
>Paul Bradbury
>Kamloops, BC
>Canada
>
>
>
>Jo-Ann Bader, Ms. wrote:
>
>  
>
>>Good Morning Everyone,
>>
>>I am looking for a stain for Collagen without a background stain.  I have tried the Aniline blue from the Masson's Trichrome with unsatisfactory results.  
>>
>>Jo-Ann Bader
>>
>>
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>> 
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