X-gal antibody

[Joannak]

Hi,

I do not know if this question was raised before, but I am looking for a good X-gal antibody (I'm working with murine embryos) that works on paraffin sections.

I tried to do X-gal whole mount staining of murine embryos (E15.5 and older) but  the penetration of staining solution was limited.  Did anyone of you tried to re-stain mouse embryo paraffin sections with X-gal antibody? Does it work?

Or do you have a good protocols for  X-gal whole mount (for older murine embryos)?

Thanks for any hint!

Joanna

[ImmunoNYC]

I have used many X-Gal abs all of which work decently however, there are a multitude of problems in level of detection. That being said, I never tested any on paraffin. If interested, I can dig up some catalog #s, let me know.

For older embryos I snap freeze them and then cut fresh 12-15um frozen sections followed by X-Gal staining ... did you try that?

[Joannak]

It would be great if you can give me some suggestions, which antibody is worth of trying.

I do not freez embryos, first I wanted to do the whole mount staining, but as far as I see it will be better to cut them first and then to stain. I am little bit afraid of making cryosections. I have heard that during cutting you can 'create' many artefacts and for chcecking the morphology is better to make paraffin sections first.. What is your oppinion about that?

Thx,

J.

I have used many X-Gal abs all of which work decently however, there are a multitude of problems in level of detection. That being said, I never tested any on paraffin. If interested, I can dig up some catalog #s, let me know.

For older embryos I snap freeze them and then cut fresh 12-15um frozen sections followed by X-Gal staining ... did you try that?

[ImmunoNYC]

It would be great if you can give me some suggestions, which antibody is worth of trying.

I have used antibodies Cortex, Biogenesis and Kappel, all on frozen sections - all work about the same. Please note, they will only work well if your level of beta-galactosidase is very high. I have not tried them on paraffin.

I do not freez embryos, first I wanted to do the whole mount staining, but as far as I see it will be better to cut them first and then to stain. I am little bit afraid of making cryosections. I have heard that during cutting you can 'create' many artefacts and for chcecking the morphology is better to make paraffin sections first.. What is your oppinion about that?

Believe me, you are right. You need to section the older ones to get good staining b/c of penetrance issues. But DO NOT be afraid of cryosections. Why not do regular X-gal on cryosections? We do it here every day with remarkable success both from glutaraldehyde perfused animals as well as fresh frozen organs sectioned and post-fixed in gluataraldehyde prior to staining. You will not create artifacts if you know what to do when cutting. And any artifacts you can create are just as easy to do in paraffin sections if one is not familiar with preparation and sectioning techniques. Paraffin sections will give you more robust histological detail but when done right cryosections can be just as beautiful and the X-Gal enzymatic detection is SO MUCH MORE SENSITIVE than anything you can ever do with a beta-Gal antibody. Trust me, go with the cryosections with X-Gal staining.

[leni2004]

rabbit anti-b gal from molecular probes/invitrogen works on paraffin sections