We're using diaminobenzidine (DAB) to visualize staining on slide-mounted paraffin-embedded mouse embryonic tissue. Often, large parts of the tissue won't stain, like a boundary is just drawn through the tissue. The unstained part is often, but not always, seen in the same area across all sections on a slide. However, we don't have this issue with fluorescent secondaries (suggesting an issue with the secondary, ABC or DAB binding) or in fetal/adult tissue (with same DAB reagents).
Has anyone else experienced this issue or have any suggestions for ways to address it? We would like to keep using DAB on this tissue if possible.
Hello Karen.
Cannot make suggestions too much due to the limited info provided, however, there seems to be mixed issues caused by 1) possibly improper deparaffinization procedure and its following clearing process, and 2) dissolving DAB-related pH issue.
1) To solve the 1st issue, my suggestion is to fix intact whole tissues (embryo or neonate or adult), followed by cut the incompletely fixed tissues into half. Change enough amount of fixative 3~4 times for another 24h. Specially brain tissues under the skull with many membranes need attention.
2) Make sure proper pH to dissolve DAB maximal, re-pH adjustment after HCl or NaOH addition and give enough time to allow pH equilibration in the tissues between any solution changes (This could be achieved by several washings with enough sol each time).
3) Use deaerated solutions to get correct pH
Best wishes.
Thank you for these suggestions, I will look into the pH of the DAB.
Our embryos are gestational day 9 - 10, so it is not easy to cut them while keeping the tissue intact. We do have success with fluorescent secondary antibodies, the penetration and staining is consistent across the tissue. The lack of staining only occurs with DAB and only in embryonic tissue. Even the fetal tissue (gestational day 17) stains well. Based on this, I agree that it likely has something to do with the DAB step.
Hi, Karen.
If the early embryos have huge anterior brain and abdominal cavities with full of fluid, it would happen unlike late embryos or neonates wit much less fluid. This may cause much slower diffusion in and out of the fluidal cavity, during fixation and washing procedure, leading to delayed exchanges of electrolytes, molecules and permeability of the thin, but tighter cell-cell contacts to ensure they are not mixed each other between the inside and outside of the embryos. The barrier only allow transport through apical and basal membrane transporters. These structural and fluidal complexes probably cause different pH or even different degree of fixation in certain tissues. Try 0.02% (v/v) Triton X-100 in your preferred pH buffer, 0.02% Triton X-100 in buffer for 5 min at RT. after the fixation and washing the remaining fixatives.
Best wishes.
mostly your problem related with in adequate time for de parafinization in addition that the cold xylin could not work properly
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