Preparation question, not microscopy related

[Farid Jalali]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello All,

Does anyone have experience storing coverslips that have been fixed with freshly prepared 2% paraformaldehyde for long term, say a month or so? I would eventually label them for indirect immunofluorescence using primary antibodies and fluorophore-conjugated secondary antibodies? This is not a standard procedure for me but circumstances require me to consider this if possible. Any advice or suggestions would be greatly appreciated.

Cheers

Farid

--
Farid Jalali MSc
Senior Research Technician- Lab Manager
Applied Molecular Oncology/ Princess Margaret Hospital
STTARR Innovation Facility/ Radiation Medicine Program
 

Toronto, Canada
 

416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite

[Masur, Sandra]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Yes - but after fixation, briefly permeabilize with dilute Triton  
X-100 and then rinse before putting back in PBS. This process will  
decrease the potential for cellular structre degradation by lightly  
fixed and still active enzymes.


On Jun 9, 2008, at 10:09 AM, Farid Jalali wrote:

> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- 
> bin/wa?S1=confocal
> Hello All,
> Does anyone have experience storing coverslips that have been fixed  
> with freshly prepared 2% paraformaldehyde for long term, say a  
> month or so? I would eventually label them for indirect  
> immunofluorescence using primary antibodies and fluorophore-
> conjugated secondary antibodies? This is not a standard procedure  
> for me but circumstances require me to consider this if possible.  
> Any advice or suggestions would be greatly appreciated.
> Cheers
> Farid
>
> --
> Farid Jalali MSc
> Senior Research Technician- Lab Manager
> Applied Molecular Oncology/ Princess Margaret Hospital
> STTARR Innovation Facility/ Radiation Medicine Program
>
> Toronto, Canada
>
> 416-946-4501 X4351 (Princess Margaret Hospital)
> 416-581-7754 STTARR at MaRS Building
> 416-581-7791 STTARR Microscopy Suite

[lechristophe]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Usually when I store my PFA-fixed cells for more than overnight I fix with 4% PFA,rinse, and put back in 0.1% PFA to avoid growth of contaminants that can occur even if stored at 4°C. An alternative is to use sodium azide, but it is simpler to just dilute the fixative and works very well in my hands.

Christophe

On Mon, Jun 9, 2008 at 16:41, Sandra Masur <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Yes - but after fixation, briefly permeabilize with dilute Triton X-100 and then rinse before putting back in PBS. This process will decrease the potential for cellular structre degradation by lightly fixed and still active enzymes.

On Jun 9, 2008, at 10:09 AM, Farid Jalali wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello All,
Does anyone have experience storing coverslips that have been fixed with freshly prepared 2% paraformaldehyde for long term, say a month or so? I would eventually label them for indirect immunofluorescence using primary antibodies and fluorophore-conjugated secondary antibodies? This is not a standard procedure for me but circumstances require me to consider this if possible. Any advice or suggestions would be greatly appreciated.
Cheers
Farid

--
Farid Jalali MSc
Senior Research Technician- Lab Manager
Applied Molecular Oncology/ Princess Margaret Hospital
STTARR Innovation Facility/ Radiation Medicine Program

Toronto, Canada

416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite