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Author Topic: toluidine blue and nitric acid in bone  (Read 11668 times)

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Offline inmuno

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toluidine blue and nitric acid in bone
« on: October 01, 2009, 06:23:37 PM »
Hi:

Does anybody know why I can't obtain any staining at all (both, bone and cartilage are totally transparent) with Toluidine blue in FFPE (overfixed) bone+cartilage samples, decalcificied with 10% nitric acid (not neutraliced before processing). The protocol is the ihcworld one, and the positive control (EDTA decalcified bone) stains really beautiful.
The problem is the Ph of the tissue??any solution for it???we are going to try more concentrated solution and much longer incubation times but I was wondering if a pretreatment with some kind of neutralization buffer or something could help??
We have tried Safranin O in the same sample and there is slight red staining in the cartilage, so some of the proteoglicans are still there...
thank you

toluidine blue and nitric acid in bone
« on: October 01, 2009, 06:23:37 PM »

Offline richard03

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Re: toluidine blue and nitric acid in bone
« Reply #1 on: October 01, 2009, 08:38:10 PM »
Are you staining mast cells?

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #2 on: October 02, 2009, 06:07:07 AM »
No,we are staining cartilage and we want to use toluidine blue for it. Our EDTA positive control Itīs ok but is very extrange, we do not obtain any color at all in nitric samples (and they are stained at the same time).

thank you

Offline richard03

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Re: toluidine blue and nitric acid in bone
« Reply #3 on: October 02, 2009, 07:53:54 AM »
Alcian Blue and Safranin O are also good for cartilage staining.

Alcian Blue Protocol
http://www.ihcworld.com/_protocols/special_stains/alcian_blue.htm

Safranin O Protocol
http://www.ihcworld.com/_protocols/special_stains/safranin_o.htm

Offline CanuckPhD

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Re: toluidine blue and nitric acid in bone
« Reply #4 on: October 02, 2009, 10:49:10 AM »
Is the Toludine Blue being washed out quickly when you dehydrate in alcohol? Or does the tissue not take it dye up at all?

Toludine blue works best at an acidic pH (about 4.0), so perhaps your tissue is too acidic after decalicification in nitric acid. Perhaps if you rinse the tissue well in water it may help or immerse sections in a neutral pH solution.

Good luck

Offline gula

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Re: toluidine blue and nitric acid in bone
« Reply #5 on: October 03, 2009, 04:59:54 AM »
I've read somewhere (?), that proteoglycans can be degraded and washed out by too acid environment. Perhaps there's too little left for staining.
I try to look up the literature ...
gula

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #6 on: October 04, 2009, 02:33:34 PM »
Hi all!

Gula,you are right, EDTA and acid decalcifiers extract the proteoglycans from the tissue (and some fixatives too) (Theory and Practice of Histological Techniques, Bancroft and Gamble) , so we have stained a serial section with Safranin O just in case, and there is positive staining,so proteoglycans are still there.
Richard, thank you for the suggestion and the protocols, we are interested in both stains just to see which is better for our tissue (I think that Toluidine could be better to obtain uniform staining with this kind of samples but...).
CanuckPhD, we will try with a neutral solution, I think that the problem is the Ph too. I have to ask if the blue washed out with alcohol or didn't take up the dye (I was not doing the technique on Friday).

Thank you all
« Last Edit: October 04, 2009, 02:36:29 PM by inmuno »

Offline gula

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Re: toluidine blue and nitric acid in bone
« Reply #7 on: October 05, 2009, 02:45:42 AM »
In Dr. Kiernan's book is described a more simple dye-solution. 0,05-0,5% toluidinblue in 1% aequeous acetic acid (pH 4). After rehydration staining for 1-5 min, wash in water, quickly dehydrating (best directly in 3 changes of abs.ethanol, or even blot dry and dehydrate in 2 changes of n-butanol), clearing and mounting.

He states, that the dye is mainly differentiated by 70% ethanol. To preserve the colour fast dehydration is necessary. Metachromasia can be observed (purple) in the intercellular matrices of cartilage.

hope this helps
gula

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #8 on: October 05, 2009, 04:03:13 AM »
Ok gula,thank you, we will try this protocol too.

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #9 on: October 05, 2009, 04:05:56 PM »
Hi:

We have rinsed the tissue in PBS for one hour and after that we have tried the protocol that Gula suggested (Dr. Kiernanīs dye solution). On Friday the tissue didn't took the dye at all , but today we have obtained positive staining, but still is not uniform (some zones of cartilage didnīt took the dye) .
We will go on with this protocol,we will try with different staining times and we will modify dehydration steps too. We have eliminate the water wash after the staining step because the dye was washed out. Appart from the sample (FFPE nitric acid decalcified sample) our FFPE and EDTA decalcified positive control is ok with this protocol.
I will post the final protocol when we finished the trial

Thank you all, your help and suggestions are always very usefull

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #10 on: October 25, 2009, 04:02:21 AM »
Hi:

We have obtained acceptable staining in nitric acid samples with gulaīs protocol but with some modifications:

- 15 minutes in Toluidine blue solution instead of 5 minutes (on 10 micron sections).
- After that we haver eliminate the liquid with an absorbent towel paper (just put the slide face down on the towel and put slight pressure on it).
- Air dry
- Dehydrate shortly in alcohol 100% directly.

The  Methacromasia is not very evident (is not totally differentiated)  but the cartilage is stained correctly. We have tried neutralizing samples before staining 1 hour with sodium bicarbonate instead of PBS but we can't see differences.

Thank you all, specially thank you Gula

Offline gula

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Re: toluidine blue and nitric acid in bone
« Reply #11 on: October 25, 2009, 01:43:44 PM »
I'm glad, that you had success. And I'm glad, that I had the right literature by the hand.
bye gula :D

Offline inmuno

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Re: toluidine blue and nitric acid in bone
« Reply #12 on: October 25, 2009, 03:23:04 PM »
Hi Gula!:

I'm very very very glad too  ;D, I hope I could reply you the favor in the future  ;) . I will try to translate to English what I always say, the important think is to know how to find the correct answer, not to know everything from the beginning.

Thanks again Gula

Re: toluidine blue and nitric acid in bone
« Reply #12 on: October 25, 2009, 03:23:04 PM »