[Histonet] help: Heidanhein's azan trichrome

From:Sasha Manhattan

Hello colleagues,   
   I am a student at the University of Hong Kong.  I am trying 
Heidanhein's azan staining to quantitate the extent of fibrosis in mouse liver.  
When I tried it for the first time, there was 
a blue colour in the tissues showing collagen deposition, which is what 
expected, but the protocol I used does not include aniline.  I only 
used 95% 
alcohol to extract azaocarmine G from collagen because our lab do not 
have aniline at the moment.  but since i got the blue colour, i think 
was ok.  but now that I repeat it with the same tissue, i dont see any 
blue colour. I thought maybe the distilled water rinse washed out the 
water-soluble aniline blue, but i repeated without washing in distilled 
water (just put in 95% alcohol and then absolute alcohol and then 
xylene clear), but there's still no blue colour.  Could you tell me 
could be wrong?

   This is my protocol:
1.deparaffination, hydration through alcohols.
2. Put slides into azocarmine G solution at 50 degrees celsius for 1 
3. Rinse quickly with dH2O.
4. Place into 95% ethanol.
5. Place into 1% acetic acid in 95% ethanol.
6. Rinse briefly in dH2O.
7. Place into 5% phosphotungstic acid for 2 hrs.
8. Place into aniline blue-orange G solution for 2 hrs.
9. Rinse very quickly with dH2O, dehydrate with 95% and 100% 
ethanol(just dip), 3 changes of xylene each of 3 minutes.  mount.

   I would be very grateful if you could just tell me what might have 
gone wrong.  There is no one for me to ask in the lab because no one 
does histology here, and I am really nervous and frustrated.  
   Thank you very much for your time and attention.

Yours sincerely,
   Mimi Kwun-nok Man 

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