7 replies to this topic

[biobunny]

hi everyone~ Need your help eagerly!!!

I found that many of the nuclei disappeared on my slide from immuno-fluorescence assay; and the rest of the nuclei which were still there seemed to be about to depart from the cytoplasm.    !!!

WHY????

Does that mean the cells had started apoptosis before I fixed them with 4% Paraformaldehyde?

Thanks very much in advance!!

[bob1]

It may be that when you put the coverslip over the cells, you pushed it sideways a bit, which can cause the cells to "smear" a bit.

[biobunny]

bob1, on 29 July 2010 - 02:10 PM, said:

It may be that when you put the coverslip over the cells, you pushed it sideways a bit, which can cause the cells to "smear" a bit.

Thanks, Bob1.   

But, if the cells just smeared a little bit, why were lots of nuclei completely gone?  

I stained the nucleus with DAPI at the same time, and there was no blue signal around the cell indicating nucleus or "smearing".

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•  Image6.jpg   73.56K   19 downloads

[bob1]

How did you permeablise the cells?  Perhaps your permeabilisation step wasn't enough to allow the DAPI in?

[biobunny]

bob1, on 29 July 2010 - 07:59 PM, said:

How did you permeablise the cells?  Perhaps your permeabilisation step wasn't enough to allow the DAPI in?

Thank you, Bob1, again!

It is better to post a photo here...

some of the nuclei were gone; and pls look at the bubble like green signal in the cytoplasm...
I am not sure why it displayed such a pattern.

In fact, before fixing the cell, I treated them with digitonin in order to permeablize cell membrane, and the cytosol/cytoplasmic contents should have been rinsed away through the holes on the membrane...

"Does that mean the cells had started necrosis or apoptosis before I fixed them with 4% Paraformaldehyde?"

Attached Files

•  Image6.jpg   73.56K   17 downloads

Edited by biobunny, 29 July 2010 - 08:13 PM.

[bob1]

Thanks for the picture, you have some nice IF there.  You definitely haven't smeared the cells and they look appropriately permeabilised.

What was the green IF for?  It may be that they are starting to apoptose, but it is hard to tell. I would have expected the cells to be a bit more rounded and in some cells the DNA to  be condensed into bodies around the nuclear membrane, before it would disappear totally.

Sorry, you have me stumped as to what is actually happening...

[biobunny]

bob1, on 01 August 2010 - 02:01 PM, said:

Thanks for the picture, you have some nice IF there.  You definitely haven't smeared the cells and they look appropriately permeabilised.

What was the green IF for?  It may be that they are starting to apoptose, but it is hard to tell. I would have expected the cells to be a bit more rounded and in some cells the DNA to  be condensed into bodies around the nuclear membrane, before it would disappear totally.

Sorry, you have me stumped as to what is actually happening...

I had done a nuclear import assay before I fixed the cells--

I used digitonin to permeablize the cell membrane, then rinsed them; theoretically the cytoplasmic contents should have been washed away; then I added some nuclear-localiztion protein to the buffer, then supplemented energy generating system so that the protein could translocate into the nucleus.

The whole process took about 1hr before the fixation; I found the loss of nuclei were much worse in samples treated with higher concentration of digitonin....

And the green signal indicates the extrogeneous protein.

Will the information above help to explain the reason?

[bob1]

From a little bit of reading, the reason digitonin is used is that it is better at dissolving the cytoplasmic membrane than the nuclear, I suspect that you have managed to permeabilise some of the nuclei but not all.