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Nuclear staining - How to permeabilise the nuclear envelope? (Dec/01/2006 )

Hi!

I need to do a nuclear staining for my experiments. I bought a Histone2B antibody which works great in western blotting but not in ICC. I thought that mybe the problem lies in the permeabilisation step. I use 0,1% TritonX100 for 15 minutes. Do you have any experience how to make it work?
Or do you know a good nuclear dye? We don't have a UV laser in our institute so DAPI won't do. I would prefer something red.

Thanks

Josephine

-josephine-

QUOTE (josephine @ Dec 1 2006, 06:31 PM)
Hi!

I need to do a nuclear staining for my experiments. I bought a Histone2B antibody which works great in western blotting but not in ICC. I thought that mybe the problem lies in the permeabilisation step. I use 0,1% TritonX100 for 15 minutes. Do you have any experience how to make it work?
Or do you know a good nuclear dye? We don't have a UV laser in our institute so DAPI won't do. I would prefer something red.

Thanks

Josephine



Propidium iodide

-dnafactory-

Can you use HE staining without Eosin step for nuclei visualization under light microscope?

-genehunter-1-

hi,

i succeded permeabilization of adherent Renca cells for NFkB cytoplasmic/nuclear staining with Triton 1% for 5 min at RT, that after fixation in PFA 4%
i did not used Triton in subsequent steps

S├ębastien

-tryptofan-

I finally succeded using PFA 4% for 10 min and permeabilization with -20 acetone for 10 min in -20

-josephine-

QUOTE (josephine @ Dec 1 2006, 06:31 PM)
Hi!

I need to do a nuclear staining for my experiments. I bought a Histone2B antibody which works great in western blotting but not in ICC. I thought that mybe the problem lies in the permeabilisation step. I use 0,1% TritonX100 for 15 minutes. Do you have any experience how to make it work?
Or do you know a good nuclear dye? We don't have a UV laser in our institute so DAPI won't do. I would prefer something red.

Thanks

Josephine


an Ab which works in Wb must not necessarily work in ICC/IHC; saponine is an alternative or add-on to triton X-100

-The Bearer-

So, back to controls....
When doing an experiment use a positive and negative control.
Use IgG as a negative and somethin g that has previously been published many times
as a positive. This will telll you if your protocol is working.
I always use 0.1% triton and this works fine. A control for nuclear staining
will tell you if your protocol is working. There are many controls yolu can use.

-mikew-

QUOTE (josephine @ Dec 1 2006, 04:31 PM)
Hi!

I need to do a nuclear staining for my experiments. I bought a Histone2B antibody which works great in western blotting but not in ICC. I thought that mybe the problem lies in the permeabilisation step. I use 0,1% TritonX100 for 15 minutes. Do you have any experience how to make it work?
Or do you know a good nuclear dye? We don't have a UV laser in our institute so DAPI won't do. I would prefer something red.

Thanks

Josephine


you could try toto 3 iodide

-Dominic-

QUOTE (josephine @ Dec 1 2006, 11:31 AM)
Hi!

I need to do a nuclear staining for my experiments. I bought a Histone2B antibody which works great in western blotting but not in ICC. I thought that mybe the problem lies in the permeabilisation step. I use 0,1% TritonX100 for 15 minutes. Do you have any experience how to make it work?
Or do you know a good nuclear dye? We don't have a UV laser in our institute so DAPI won't do. I would prefer something red.

Thanks

Josephine


Just remember if you use TOTO-3, you will have to use an RNAse. If you get RNAse...Ambion makes a good one with a couple different enzymes in them. The reason you need the RNase is because TOTO-3 is not specific for ds nuclei acids. But it does look great..and FYI it's red when imaged.

-viper-

QUOTE (genehunter-1 @ Dec 1 2006, 08:41 PM)
Can you use HE staining without Eosin step for nuclei visualization under light microscope?


yes you can - the eosin just provides a usefull referance as to where the nuclei are but i get the feeling we are talking fluorescance microscopy here so it would have to be a fluorescant dye to appear on any pictures you produced.

-Dominic-