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,