[Histonet] Masson's Trichrome Troubleshooting

[John Kiernan]

If the known positive control slides were side-by side with the study slides (which stained correctly), then there must be something wrong with the control slides, not the solutions or the steps of the method. Different fixation or inadequate removal of paraffin come to mind, but you've probably thought of those. Your study slides of heart etc were well stained, so perhaps this will be a suitable positive control for future Masson stainings.

It would be satisfying, however, to know why previously positive collagen in your sections of muscle etc failed to stain with aniline blue. Was the collagen in these sections red or not stained at all? If it was red, then the PTA/PMA had failed to displace the red dye, and a longer time in PTA/PMA is likely to be needed. If that's the case the control sections are unsuitable for staining beside your study sections. The final wash is in dilute acetic acid to prevent the removal of any bound red or blue dye, which can occur if plain water is used. You rightly examined the wet sections at this stage, an action that shows that you know what you are about, unlike many professors, postdocs, graduate students, . . .

Please let us all know if you find out why the collagen in previously good positive-control tissue became unstainable with the Masson's method in Luna's AFIP manual. 

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada   N6A 5C1
   http://publish.uwo.ca/~jkiernan/index.htm
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----- Original Message -----
From: "Ford, Judi" <judi.ford <@t> roche.com>
Date: Wednesday, August 8, 2007 15:48
Subject: [Histonet] Masson's Trichrome Troubleshooting
To: histonet <@t> lists.utsouthwestern.edu

> Hi everyone,
> 
>  
> 
> I'm hoping someone can give me suggestions on what happened to 
> my stain.
> I did a Masson's Trichrome on mouse hearts/aortas this 
> morning.  I
> followed the AFIP protocol exactly and used two different 
> control slides
> (tongue, aorta, skeletal muscle). We have previously stained control
> slides with the tissue I used this time and they worked beautifully.
> 
>  
> 
> After finishing the glacial acetic acid rinse I examined the slides
> under the scope and found that neither of the control slides stained
> with the aniline blue, but all the study slides stained 
> beautifully with
> aniline blue. I tried going back and restaining (from
> phosphomolybdic/phosphotungstic acid) the slides and the same results
> showed. I thought maybe I had them in the glacial acetic acid 
> too long
> so I cut that back to 3 minutes instead of 5 for the second round.
> 
>  
> 
> Any ideas on what could have happened? Could the control slides have
> been sitting too long as unstained slides? Could the aniline blue
> working solution need an extra punch from glacial acetic acid? 
> In the
> past I've reused aniline blue without any problems and this solution
> wasn't past its expiration date.
> 
>  
> 
> Would love to hear your thoughts......
> 
>  
> 
> Judi Ford
> 
> Palo Alto, CA
> 
>  
> 
>  
> 
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