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
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
Can you use HE staining without Eosin step for nuclei visualization under light microscope?
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
Sébastien
I finally succeded using PFA 4% for 10 min and permeabilization with -20 acetone for 10 min in -20
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
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.
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
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.
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.