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Author Topic: Tissue dissolves after DAB treatment in IHC  (Read 3544 times)

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Offline GJudith

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Tissue dissolves after DAB treatment in IHC
« on: October 01, 2012, 09:59:43 AM »
Hi there,

Iím dealing with a tissue dissolving problem during DAB development in IHC and would like to hear your opinion. I canít make any sense out of it.. :'(

Iím doing IHC of intestinal tissue (cryosections, 8Ķm, acetone fixed) in order to isolate CD68+ cells of the lamina propria by laser microdissection. The final aim is to isolate RNA for Genechip analysis.

As it is problematical to obtain intact RNA after immunostaining, Iím using a TBS buffer with a total concentration of 2M NaCl, as I found a publication which said that a 2M NaCl concentration is able to stabilize RNA as good as RNAlater (which I canít use because itís supposed to be incompatible with IHC). Iím using the 2M NaCl TBS buffer as washing buffer as well as for the antibody diluent. I develop with DAB ImmPACT (liquid) from Vector. My secondary antibody is HRP labeled and from DAKO (I tried the ABC-Kit from Vector but had the same results). I have tried both super fros plus slides as well as membrane slides.

My problem is that my tissue is sort of dissolving right at the moment I put the DAB on the section. It sort of looks like some layers are floating off the section.

I only have this problem under certain conditions:
1.   If I donít use any antibodies, my tissue looks perfectly fine
2.   If I use a secondary antibody (primary has not to be present), the tissue dissolves the moment I put the DAB on it (before that it looks totally fine, I checked with a Hematoxylin stain).
3.   If I use normal TBS (0.15M NaCl) as buffer, everything is fine (with and without antibodies)
4.   If I use 2M NaCl TBS as a buffer in all steps but wash with normal TBS before I use the DAB, the tissue still dissolves
5.   If I use 0.5M NaCl TBS as a buffer in all steps, the tissue dissolves
6.   If I use 2M NaCl TBS as a buffer in all steps, and in the end do consecutive washing in 1M NaCl TBS, then 0.5M NaCl TBS, then normal TBS, it still dissolves when I put DAB on the section.
7.   I have also tried using the 2M NaCl TBS buffer as the diluent for the chromogen concentrate instead of the provided diluent, but still my tissue dissolves.

I already contacted Vector, but they donít have any experience with this phenomenon. The very last idea I have is to try a different substrate as AEC.

Iím totally at a loss and donít know, what to do, I really hope, you can help me!

Thank you so much,
Judith

Tissue dissolves after DAB treatment in IHC
« on: October 01, 2012, 09:59:43 AM »

Offline gula

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Re: Tissue dissolves after DAB treatment in IHC
« Reply #1 on: October 01, 2012, 11:10:46 AM »
I have no real answer, but I think you create a kind of solvent with many Na-Ions and the organic solvent of the DAB. It reminds me of the use of SDS for desintegration of tissue. Find out the solvent of the DAB.
Perhaps the reagenses together can interfere with the cellmembranes, and additional concure to the binding sites on the slide.
Perhaps you can use DAB powder (tablets) solved in your buffer.

good luck
gula

Offline GJudith

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Re: Tissue dissolves after DAB treatment in IHC
« Reply #2 on: October 08, 2012, 05:52:14 AM »
Hi Gula,

thank you for your help! I will try using another DAB and let you know if it worked.

Thanks,
Judith

Re: Tissue dissolves after DAB treatment in IHC
« Reply #2 on: October 08, 2012, 05:52:14 AM »