[Histonet] Re: Neurofilament - LFB combo -- problems

[Theresa (Teri) Johnson]

Karen writes:
To anyone who performs a NF-LFB (+PAS) combo stain using a Ventana
Benchmark:

I have been trying to stain a nerve (cross and longitudinal section) and it keeps falling off of the slide after the neurofilament stain.  It's been cut onto a charged slide and has been baked 20 minutes prior to IHC staining.  After IHC staining, we have rinsed in Dawn-water per protocol; by then the longitudinal section is peeling off the slide.  I tried skipping the Dawn rinse and have gone to straight 95% alcohol and
1 of 4 sections survived on the slide (others fell off).

Tissue problem - the nerve is well myelinated and fatty.  If I drop the water bath temp too low, the nerve doesn't flatten well; if the temp is too high, it's shredding and spreading.

Has anyone out there had this problem and resolved it?  Or do you have a protocol that might have avoided it completely?

Any suggestions would be appreciated.

Hi Karen,

I don't know if any of this will help you, but here are a few suggestions. You can treat this tissue like we would brain or bone, after the water is drained off the section, lay the slides flat, tissue side up, on a slide warmer at lower temp, maybe 37-45 degrees C. You can try overnight and see if this helps. You can also try using Haupt's adhesive treated slides, they use this for plastic sections and bone and it tends to work pretty well. You can buy it commercially from Dorn and Hart, or you can make your own. The recipe is on the internet. Maybe use a combination of both?

Try baking it a little longer too, 1-2 hours at 60 degrees if the above doesn't work.

I know how frustrating this can be! Good luck.

Teri Johnson, HT(ASCP)QIHC
GNF Histology Lab Manager
Genomics Institute of the Novartis Research Foundation
858-332-4752