[Histonet] Harris Hematoxylin troubleshooting

[Delatour Benoît]

Dear all,

I recently tried to do a H&E stain on mice brain frozen sections using a 
protocol I sucessfully used with paraffin sections. Staining is very bad 
with frozen sections, showing awful uneven labeling (i have just posted a 
PDF file on http://www.histonet.org/ with some horrible pics ; the filename 
is HE-pics.pdf).
Here are some details:
-Brains were fixed in buffered formaldehyde 10%, then cryoprotected with 
DMSO-glycerol overnight and cut on a sliding microtome (40µm free floatting 
sections). Sections were then mounted on Superfrost+ glass slides, 
dehydrated overnight at 40°C and then processed for H&E staining.
-These sections can be perfectly stained using thionin, cresyl violet, 
Vector nuclear fast red etc... They can also be processed with success for 
IHC, histochemistry etc...
-My protocol is very (too?) simple: rehydratation in tap water - staining 
in Harris H. (filtered) for 2-4 min - tap water - acidic alcohol ...
-My H&E attemps made use of the Harris hemtoxylin from (c)Roth (see 
http://www.fr.carlroth.com/catalogue/catalogue.do;jsessionid=D750394EC8EA27E19AB00DEE85E744BA?id=6284&favOid=000000000002812b00060023&act=showBookmark&lang=en&catId=FR)
-The problem does not come from dewaxing as... brains were not embedded in 
paraffin!
-The problem does not seem to come from to short rehydratation as 20 min 
under tap water before hematein does not change anything.
-I have mounted sections on glass slides using gelatin-PBS or distilled 
water : does not make any change.
-I have increased "incubation" times in the Harris solution (from 2 to 15 
min): always uneven stain.

All suggestions/comments are welcome. Thanks!

Dr. B. Delatour


_______________________________

B. Delatour
Laboratoire de Neurobiologie de l'Apprentissage,
de la Mémoire & de la Communication,
NAMC, CNRS UMR 8620, Bât 446
Université Paris-Sud
91405 Orsay Cedex, FRANCE
Email benoit.delatour <@t> ibaic.u-psud.fr
Web http://www.namc.u-psud.fr