[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! (Maria Mejia)

[Steven Weston]

Maria,
This may sound simplistic but I find that if I have problems such as this the first thing I do is try a new batch of the staining reagents. If you haven't had problems before then something must have changed either in your protocols or your reagents. It could be that your heat retrieval reagents are too old or contain detergents that remove some of the proteins you are looking for. Some of the proprietary heat retrieval reagents that allow you to heat retrieve without having to dewax  by going through xylene have been shown to change the nuclear staining pattern and create what appear to be nuclei that are full of vacuoles.
Also if the celloidion is not completely removed during your staining it may be stopping any of the higher molecular weight stains from penetrating the cells. Try leaving your sections in acetone for a number of changes to ensure full removal of the celloidion.
Regards
Steve Weston
University of Tasmania
Breathe-Well CRE
Lab Manager
0408990859




University of Tasmania Electronic Communications Policy (December, 2014).
This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise.