[Histonet] LacZ and cryosectioning

[S.J.Ainsworth <@t> bsms.ac.uk]

The chick hindlimbs are removed and washed in chick ringer's solution (a salt solution to wash off and debris) then fresh frozen with cryo-m-bed. On the sections we fix with 2% PFA for 10mins, but the problem is arising before the sectioning. I am sure that the stain should be stronger as the staining on my positive controls that are frozen is massively reduced. Does that make any more sense?

Thanks for your help

Sophie

Sophie Ainsworth 
Brighton and Sussex Medical school 
University of Sussex 
Falmer 
East Sussex 
BN1 9PX 
Tel: #44 1273 877886 
Fax: #44 1273 877884 

-----Original Message-----
From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu] 
Sent: 08 January 2009 18:34
To: Ainsworth Sophie; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] LacZ and cryosectioning

We do X-gal on snap frozen tissue all the time, so I can try to help. Can you give more specifics about your protocol? Your tissue is fresh frozen, correct? In OCT? Specifically what fixative are you using on your sections and for how long? Are you using Ca2+ and Mg2+ free PBS? Are you positive your tissue should be a strong stainer - if not do you have a sure-fire positive control?

Andrea

-----Original Message-----
From: S.J.Ainsworth <@t> bsms.ac.uk

Date: Thu, 08 Jan 2009 17:17:41 
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] LacZ and cryosectioning


Hi,

 

I’ve been having some trouble x-gal staining some cryosections of chick hindlimb tissue. I’ve been cutting 15 µm sections then x-gal staining (using a protocol from another lab that should work) and counter staining with nuclear fast red. The sections look good with the nuclear fast red, but the x-gal staining only shows up as very faint speckling at x40. I routinely x-gal stain whole chick embryos then wax section and the blue of the x-gal stain is always very bright (both on the embryo before any tissue is removed for sectioning and in the wax sections). To try and find out if the problem was occurring before or after sectioning I defrosted some limbs (that had not as yet been cryosectioned) and x-gal stained them using the method that I use routinely, they showed none of the blue staining I would expect. Has anyone heard of snap freezing knocking down the activity of the β-galactosidase enzyme or is able to suggest anything I could try to solve this, please?

 

Thanks

 

Sophie

 

Sophie Ainsworth 
Brighton and Sussex Medical school 
University of Sussex 
Falmer 
East Sussex 
BN1 9PX 

Tel: #44 1273 877886 
Fax: #44 1273 877884