Trisha

I've used Fisher's "Super Frost Plus" slides for paraffin sections (coat
them w/ poly-L-lysine for frozen sections I've learned recently from this
Histonet) and only had trouble with sections falling away doing the BrdU
assay.  If you're sialyzing your own slides you may be spending more time
and money than you need to be.  Super Frost Plus slides are more expensive
but you can avoid the other hassels.

I don't know what sort of assay you use but the assay I follow uses a pepsin digest (pH 2 or whatever the HCl gives
you) followed by incubation in 4N to 6N HCl.  The pepsin digests away the
chromatin-associated proteins so the HCl incubation can denature long
enough stretches of DNA to expose sufficient BrdU to show up in your
assay.  While the pepsin is freeing the DNA it is also digesting proteins
throughout the tissue sample that are the sticking agents holding the
tissue onto the slide.  The HCl will change the charge, which is what
makes the tissue stick, on all charged
groups.

So, if you're doing a similar BrdU assay, try shortening the pepsin digest
time and using a lower [HCl] and incubation time since the pH will also
alter the charge on the proteins.  If you're not doing a similar BrdU
assay, ignor all of this and delete immediately.

yours hoping for a stormy Monday



John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849


On Wed, 11 Aug 1999, P. Emry wrote:

> Hi Netters
> 
> I am having the problems of tissue coming off the slides durning Brdu
> staining.  I am not the one doing the staining, but I'm the one cutting
> the sections.  Is there a etoh step in Brdu that can be avoided so that
> the tissue will stay on?  Any other modification that will help?
> 
> I ordered my silane from Sigma then saw that Surgipath only makes it up
> when you order it because it has a shelf life of 3 months.  Any thoughts
> on this or experience?
> 
> Trisha   
> On Wed, 11 Aug 1999, John C. Dennis wrote:
> 
> > Richard
> > 
> > Are you using any EtOH in treating your tissue before you mount the
> > sections on slides?  
> > We were having a similar problem just the other week.  Our frozen
> > sections were coming off the
> > slides poly L lysine or no.  The tissue had been fixed but held in 70%
> > EtOH until sectioning.
> > Once I stopped exposing the tissues to EtOH the sections stopped coming
> > off the slides.
> > 
> > John Carroll Dennis
> > Anatomy, Physiology, and Pharmacology
> > 109 Greene Hall
> > Auburn University, AL  36849
> > 
> > 
> > On Wed, 11 Aug 1999, Richard G. Lea wrote:
> > 
> > > To whoever can help,
> > > 
> > > I am attempting to carry out in-situ hybridisation on sections of tissue
> > > which have been cultured for 24 hours.  The control non-cultured tissues
> > > are fine but after culture the tissue sections fall off the slides
> > > during the in-situ protocol.  We have tried TESPA and double coated
> > > gelatin, PLL slides however the problem remains.  How can I stop these
> > > sections coming off the slides - any suggestions gratefully received.
> > > 
> > > Richard Lea
> > > -- 
> > > Richard G. Lea PhD
> > > Senior Research Scientist
> > > Division of Nutrition, Pregnancy and Development
> > > Rowett Research Institiute
> > > Greenburn Road
> > > Bucksburn
> > > Aberdeen AB21 9SB
> > > 
> > > Tel:  44(0) 1224 712751 x 2319
> > > Fax:  44(0) 1224 716622
> > > 
> > 
> > 
> > 
> 
> Trisha  
>