I try to enumerate DAB-stained CD4- and CD8-T-lymphocytes in 10 µm
cryostat-sections of porcine ileum.
I counterstained the sections with Mayer`s hematoxylin (10% aqueous
The counterstaining was performed as follows:
1. Hematoxylin for 15 sec
2. short dip into ethyl alcohol/ 0,1% HCl (regressive step)
3. 5 min in tap water (for blueing)
4. One dip into double-distilled water
5. in each case 5 minutes:
* 50% ethyl alcohol
* 70% ethyl alcohol
* 99% ethyl alcohol
* isopropyl alcohol
6. 20 min in xylene
7. Mounting with Entellan (permanent mounting medium,non-aqueos)
Unfortunately, the contrast between DAB and the hematoxylin-counterstaining is too weak because the DAB-stained cells appear to be brown and hematoxylin produces a dark violet.
With this result it is not possible to enumerate the positive stained T-cells. With prolonged colouring in hematoxylin I get nicely distinguishable nuclei but the contrast to DAB gets even poorer.
Does anyone have an idea how to change the hematoxylin-protocol to achieve a better contrast? It would be nice if there is a way to turn hematoxylin from dark violet into a clear blue that I already observed in paraffin sections.
I`ve already tried forced blueing in ammonia water, without any mprovement.
Please tell me about your experiences with hematoxylin-counterstaining and DAB.
Or is it the best solution to try another dye like methyl green??
Thanks for your help,
Simone from Germany