5 replies to this topic

[seanisaken]

Hello,
Does anyone know a good protocol for IHC within nucleus.  I am looking to stain animal cells.  Fixative choice?  Permeabilization?  etc.  Thank  you.

[bob1]

stain with what?  for which proteins/molecules? There are a lot of things you can look at in the nucleus, and pretty much any standard IHC protocol should work for abundant proteins.

[seanisaken]

bob1, on Dec 8 2009, 03:25 PM, said:

stain with what?  for which proteins/molecules? There are a lot of things you can look at in the nucleus, and pretty much any standard IHC protocol should work for abundant proteins.

It is a newly identified actin-reorganizing cytoplasmic protein.  I am also wondering whether it is functional in the nucleus.  I don't know how abundant it is in the nucleus (working on doing nuclear westerns).  I was wondering whether 4% para for 15 minutes, and 0.2% triton for 10 minutes work.
Thank you.

[bob1]

Cell culture or tissue?

If cells - I use ice cold 2% PFA for 15 minutes, and lots of washes in PBS. For tissue I would go with the full 4% overnight in the fridge.  0.1-0.5% triton X100 in PBS or TBS is fine for permeabilizing cells, you can't really over-permeabilise, but 10-20 min should be enough.  I would block the cells (0.5-1 hour) before adding primary antibody, wash 3-4 times in PBS or TBS with 0.1% tween 20 for 5 min each, then add secondary, more washes, etc.

Antibody steps should be done in a humid environment so as to minimise evaporation from the solution.

If you lack a nuclear marker (Hoescht or DAPI) - Propidium Iodide will work with a red channel (green excitation).

[seanisaken]

bob1, on Dec 8 2009, 03:57 PM, said:

Cell culture or tissue?

If cells - I use ice cold 2% PFA for 15 minutes, and lots of washes in PBS. For tissue I would go with the full 4% overnight in the fridge.  0.1-0.5% triton X100 in PBS or TBS is fine for permeabilizing cells, you can't really over-permeabilise, but 10-20 min should be enough.  I would block the cells (0.5-1 hour) before adding primary antibody, wash 3-4 times in PBS or TBS with 0.1% tween 20 for 5 min each, then add secondary, more washes, etc.

Antibody steps should be done in a humid environment so as to minimise evaporation from the solution.

If you lack a nuclear marker (Hoescht or DAPI) - Propidium Iodide will work with a red channel (green excitation).

Thanks bob!  Slightly different than regular IHC.  So, I should be fine using DAPI as nuclear marker?  Btw, I am staining cells.

Edited by seanisaken, 09 December 2009 - 09:29 AM.

[bob1]

DAPI is excellent as a nuclear marker, of course it only works in fluorescence, but there are plenty of proteins that could be used if doing chromatogenic staining.

The steps are more or less the same as doing IHC or staining a western blot.  I can pm you my protocol if you like.