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Author Topic: Phosphomolybdic acid in the Masson Trichrome stain  (Read 12343 times)

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Offline NScott

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Phosphomolybdic acid in the Masson Trichrome stain
« on: December 15, 2013, 09:18:11 AM »
Can hydrochloric acid substitute for phosphomolybdic acid in the Masson Trichrome stain?

I am trying to find out why or why not this would work. And I can't find any information in my book or on the internet.

Anything would help. Thank you!

Phosphomolybdic acid in the Masson Trichrome stain
« on: December 15, 2013, 09:18:11 AM »

Offline gula

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Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #1 on: December 16, 2013, 01:40:46 PM »
Phospmolybdic acid is a polyacid with high molecular weight. there are diffrent theories about the background. One is the "Rate of staining" theory from Baker. The smallest dye is the first at the binding sites and will stain the whole tissue, then the next-bigger follows and displaces the smaller one in the "loose" areas like collagen. Baker (1958) ranges the dye-sizes Biebrich Scarlet, then !PMA! and the anilinblue (as a conglomerat). So here it is important, that Baker looks at PMA as a colorless dye. In fact the molecular weight of PMA (1825) is much higher than that of anilinblue.
Onother theory from Puchtler says, that PMA works as a bridge between collagen and anilinblue. Collagen is a basic glycoprotein and binds acid dyes. Puchtler says that PMA has a bigger affinity to collagen than Biebrich Scarlett and displaces it. But afterwards it still sticks on the collagen, when anilinblue reaches it. Anilinblue has basic aminogroups and can be seen as amphoteric dye. Puchtler says, anilinblue binds over the basic sites onto the PMA on the collagen.
Paul Prento made some experiments with VanGieson-stain. He came to the result, that it is a matter of binding-type (electrostatic, hydrogenbonds, hydrophobic interaction) which substrate "likes" which dye.
PMA can be used as single reagens before the dyes, "between" the dyes or as mixture within the dye-solution. If you put it before the dyes, you can decrease the red colour and enhance the blue color. So the cytoplasmic dye is hampered to bind, but the collagen-dye binds better. With Masson-Trichrom you can increase the brightness of the cytoplasmic colour by prolonging the PMA. Used in the dye-mixture it acts similar, the longer the brighter.

So I think this special characteristics cannot be copied by hydrochloric acid. But experiments showed, that PMA is not necessary at all to give the quite same results. (?) Probably the truth is a mixture of all theories.

P. Prento 2001; A contribution to theory of biological staining based on the principals of structural organisation of biological macromolecules (Biotechnic and Histochemistry)
(16)   Puchtler H., Isler H; The Effect of phosphomolybdic acid on the stainability of connective tissue by various dyes; J. Histochem.Cytochem 1958
(32)   Prento P., Van Gieson‘s picrofuchsin. The staining mechanisms for collagen and cytoplasma, and an axamination of the dye diffusion rate model of differential staining. Histochemistry, 1993 Feb;99(2):163-74
(35)   Horobin RW, Biological staining: mechanisms and theory; Biotech Histochem. 2002 Jan; 77(1):3-13
(21)   Silverman L, Glick D., The Reactivity and Staining of Tissue Proteins with Phosphotungstic Acid, J. Cell.Biol. 1969

tell me your results of research.
gula

Offline NScott

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Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #2 on: December 16, 2013, 09:46:52 PM »
That does help me toward the right direction, thank you.

I think ill experiment tomorrow.

I would like your opinion though.

When substituting the phosphomolybdic acid for hydrochloric acid, should I:

replace the whole phopsomolybdic/phosphotungstic acid solution with hydrochloric acid?
or just replace the phosphomolybdic acid in the solution?
Would it be worth it to try both? And what do you think the concentration of the HCl acid should be?

thanx much!

« Last Edit: December 19, 2013, 11:37:38 AM by NScott »

Offline gula

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Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #3 on: December 17, 2013, 11:15:26 AM »
PMA and PTA have more or less the same characteristics. You can use PTA allone and I think, the results are quite the same as with PMA/PTA mixture.
I don't know what will happen if one mixes PTA and HCl! That's a question for a chemist, but I would't try it.

You can try HCl alone, perhaps 1%, and after the decolorisation-step go forward with dehydration and coverslipping. PMA/PTA destains the collagen-fibers and the result is red muscle (cytoplasm) and colorless collagen. If HCl has a similar effect, this could prove it.
For us it looks like destaining, but in real PMA/PTA binds to collagen and displaces the dye. I don't think, that HCl sticks on the fibers.
You can also skip PMA/PTA at all and look at the result. It will be less bright, but cytoplasma is red and collagen is blue.


good luck
gula

Offline NScott

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Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #4 on: December 18, 2013, 11:46:50 PM »
I only ran one stain of each. But in my short sighted experiment the substitutions were 1% 3% and 5% HCl. Results were undeniably less desirable with HCl. It appears that minor adjustments, to concentration and procedure, can be made to get readable results. Not satisfactory but readable. The HCl(1%,3%,5%)solution enabled too much aniline blue . And so the final result was an over staining of blue.


I think I would rather keep my chemicals in stock then have to resort this.
« Last Edit: December 19, 2013, 11:44:03 AM by NScott »

Offline gula

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Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #5 on: December 19, 2013, 02:34:10 PM »
This could be an effect of too low pH. If the isoelectric point of all proteins is much higher than the pH, they all stain in the same manner. The difference makes the point.
The pH of the anilinblue staining solution is 2,5. The HCl should be similar, I assume.
bye
gula

Re: Phosphomolybdic acid in the Masson Trichrome stain
« Reply #5 on: December 19, 2013, 02:34:10 PM »