5 replies to this topic

[pmaj]

Hi all,

I am doing  the immunofluorescence assay, and am following a protocol which calls for fixing the cells before treatment. I fixed the cells with 4% formaldehyde for 8 min at room temp. and I saw a lot of cell debris after the fixation step. Was the formaldehyde treatment too toxic and the cells died?

Am a little disappointed.

I would really appreciate if someone could shed some light on this.

Sincerely,

Pmaj

[bob1]

Cells that have been fixed are not alive - the fixation process crosslinks the proteins so that they don't move, this is exactly what it is supposed to do so that the structure of the cells is preserved.  This is the major reason why we don't use formaldehyde in food preservation, residual formaldehyde will fix your throat as you are eating.  

What are your cells grown on?  If they are on glass; a lot of cells have problems attaching to glass, you can try coating the slides or coverslips with gelatine or poly-lysine to help the cells attach.

It could be that you were too rough with the addition of the formaldehyde and/or washes - add it slowly down the side of the well/chamber you are using to culture your cells.

Edited to add:  Your formaldehyde is buffered isn't it?  You made it up if an isotonic solution such as PBS or TBS?

Edited by bob1, 19 January 2010 - 03:47 PM.

[pmaj]

bob1, on Jan 19 2010, 06:45 PM, said:

Cells that have been fixed are not alive - the fixation process crosslinks the proteins so that they don't move, this is exactly what it is supposed to do so that the structure of the cells is preserved.  This is the major reason why we don't use formaldehyde in food preservation, residual formaldehyde will fix your throat as you are eating.  

What are your cells grown on?  If they are on glass; a lot of cells have problems attaching to glass, you can try coating the slides or coverslips with gelatine or poly-lysine to help the cells attach.

It could be that you were too rough with the addition of the formaldehyde and/or washes - add it slowly down the side of the well/chamber you are using to culture your cells.

Edited to add:  Your formaldehyde is buffered isn't it?  You made it up if an isotonic solution such as PBS or TBS?

Hi Bob,

Yes my formaldehyde was not buffered:-(. The cells were attaching fine and I was being extra gentle to them. Thanks for the input. I really appreciate it!

Pmaj

[bob1]

Try buffering your formaldehyde - it will stop the cells exploding from osmotic pressure while they are fixing.  I personally would use 2% formaldehyde on ice for 10 minutes for cells on glass.

[aimikins]

what type of cells?

I used to do IF on NHEK cells. the protocol was to fix for 1 min, 4%. over-fixing can also be a problem and cause staining artifacts.

[pmaj]

aimikins, on Jan 20 2010, 08:26 PM, said:

what type of cells?

I used to do IF on NHEK cells. the protocol was to fix for 1 min, 4%. over-fixing can also be a problem and cause staining artifacts.

Hi Aimikins,

I fixed the cells for 5 min at room temp. Is it better to fix them on ice?

Pmaj