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Post subject: Double staining with Ziehl-Neelsen and DAB
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Hello,
does anyone know how to double stain involving acid fast staining. I tried a DAB stain with a biotinylated antibody followed by acid fast staining but can't see any positive mycobacteria (maybe overlayed by the DAB?)
So, I would need to use acid-fast staining and also an antibody for eukaryotic cells.
cheers
Trilby |
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 Fri Jul 23, 2010 3:10 am |
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Have you done a ZN alone and verified the presence of mycobacterias? Usually it's said, that if you see one bacteria there should be lots of. So they should also be present in the next slice. I find it very hard to see small red acid fast bacili wether counterstained or not.
If you have the possibility for immunofluorescence, the Rhodamin-staining for AFB would be a possible alternative.?. - Never done personally!
good luck
gula |
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| Fri Jul 23, 2010 7:05 am |
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Post subject: Good points by gula.
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I add a Q or 2:
what tissue/tissue prep?
Are you wanting to stain for AFB and also immunostain for ANOTHER target?
Sorry, it is not clear to me.
Please let us know more details.
For eg, Pwax/frozen sections?
What's the other Ab?
Please give more details....thanks!
Confused Carl |
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| Sat Jul 24, 2010 4:20 pm |
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Post subject:
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Thanks for the reply and sorry for the confusion,
here are some more details.
tissue: lymph node slides from Balb/c (10um thick) frozen sections and fixed with 4% PFA
yeah I have done single staining (acid fast with Meyer's haematoxylin) alone which works beautifully, also have done the DAB stain alone (biotinylated CD169 antibody + Strep-HRP + DAB + meyer's) which also works nicely.
However, when I combine the two (again with meyer's as contrast) I can see the DAB stain but not the AF stain.
@gula: never done the rhodamine stain.
cheers |
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| Mon Jul 26, 2010 1:05 am |
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Post subject: Trilby..
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Which do you do 1st?
Acid Fast/Immuno?
reverse them?
NB: MAyer's Haemalum 
NB2: forget the Mayer's.
Have a look down the scope....if you see nothing, fair enuff.
We move on.
Mayer's will only ever counterstain all nuclei, Trilby.
It is important but, to get your staining working, Hx is not important.
My apologies if I got your Post all wrong.
Old geezer,
Carl |
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| Tue Jul 27, 2010 7:37 pm |
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Post subject:
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the AFB are at the same site as your DAB-stain? Then it could really happen, that the brownish DAB and the reddish AFB have too little contrast to be descriminated.
Is it very important to do the doublestain? Wouldn't it be proofed well enough on consecutive slides?
Another way would be to do two IHC stains with an antibody against mycobacteria. Perhaps with a contrasting blue on red? - just collecting thoughts...
But doublestaining of antigens at the same site is also not easy. Could end up in the same problem.
The IHC staining should be faint enough to see the AFB but strong enough to be positiv. - shortening DAB-incubation?
good luck
gula |
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| Wed Jul 28, 2010 5:27 pm |
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Post subject:
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thanks for your help and ideas! I tried some different things, but the double staining still wont work, will go for consecutive cuts  as both DAB and AF alone work beautifully. |
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| Thu Jul 29, 2010 5:49 am |
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