15 replies to this topic

[lormanti]

Hi,
I have been growing some normal human cells (M10) on coverslips placed in small petri s and using a very basic protocol to fix them, i.e. aspirate medium, fix them @ RT in 3.7% formaldehyde (made by diluting 37% formaldehydein PBS), remove fixative after 20 min. Unfortunately after drying out, crystals form across the slide. I have then washed my slides a few times in PBS after the fixation step and this seems to reduce crystal formation but to eliminate it. Yet, the protocol I was given did not mention any post-fixation wash. How could I avoid these crystals? Could it be the drying I am getting wrong? Thanks!

[bob1]

The crystals are salt from the PBS.  Why do you need to dry them?  You can dehydrate the coverslips in graded ethanol or methanol.

[memari]

or maybe crystals are salt (Paraformaldehyde) from the 37% formaldehyd.
A small amount of stabilizer, such as methanol, is usually added to 37% formaldehyd to limit oxidation and polymerization to Paraformaldehyde.

Paraformaldehyde (PFA) is the smallest polyoxymethylene, it is the condensation reaction product of formaldehyde with a typical degree of polymerization of 8–100 units. Paraformaldehyde forms slowly in aqueous formaldehyde solutions (formalin) as a white precipitate, especially if stored in the cold. Formalin actually contains very little monomeric formaldehyde; most of it forms short chains of polyformaldehyde. A small percent of methanol is often added as a stabilizer to limit the extent of polymerization.

https://en.wikipedia...ki/Formaldehyde
https://en.wikipedia...araformaldehyde

-----
Babak

Edited by memari, 25 July 2012 - 09:10 PM.

[lormanti]

Ok Guys, Thanks for your advice, I'll try that.
Take care.
Lorenzo

[fraffly]

Just out of curiosity, why do you dry them ? And what are you doing with them afterwards ?
I've done immunofluorescence for conventional & ElectronMicroscopy. What we usally did was, dip the slips shortly in dest.water to get rid of the salts and if you need them dry, hold a corner of the slip on a tissue to suck off the water.
If they're used for immunofluorescence afterwards I'd recommend to use a quenching solution (e.g. 50mM Ammoniumchloride in PBS) after the fixation to reduce background and false positives from free aldehyde groups, which can bind to your antibodies.

[Member2]

I am also experiencing the same problem. I am fixing Hela cells using 4% paraformaldehyde and then washing the coverslips with PBS. However when I mount the coverslips with fixed cells on the glass slide, after 2-5 minutes my coverslip starts drying up. I can see some crystals when I observe it under microscope. Could anyone please suggest why this is happening and how to get rid of it?

[bob1]

Member2, on 11 November 2012 - 12:44 PM, said:

I am also experiencing the same problem. I am fixing Hela cells using 4% paraformaldehyde and then washing the coverslips with PBS. However when I mount the coverslips with fixed cells on the glass slide, after 2-5 minutes my coverslip starts drying up. I can see some crystals when I observe it under microscope. Could anyone please suggest why this is happening and how to get rid of it?

Which surface are the crystals on (open to the air side or the side with the cells)?

[Member2]

bob1, on 11 November 2012 - 11:11 PM, said:

Member2, on 11 November 2012 - 12:44 PM, said:

I am also experiencing the same problem. I am fixing Hela cells using 4% paraformaldehyde and then washing the coverslips with PBS. However when I mount the coverslips with fixed cells on the glass slide, after 2-5 minutes my coverslip starts drying up. I can see some crystals when I observe it under microscope. Could anyone please suggest why this is happening and how to get rid of it?

Which surface are the crystals on (open to the air side or the side with the cells)?

The surface of coverslip with cells mounted on the slide (coverslip in contact with slide)

[bob1]

What are you mounting the coverslip in?

[Member2]

bob1, on 15 November 2012 - 11:54 AM, said:

What are you mounting the coverslip in?

I am not using any mounting media because I am just imaging the autofluorescence of my nanoparticles (cells treated with nanoparticles) under confocal microscopy (I am not doing any immunochemistry assay). I am intrested in knowing if my nanoparticles internalized into the cell or not ? So what I do is, I fix the cells with paraformaldehyde grown on coverslip, wash with 1XPBS and put the coverslip on slide (with cell side of coverslip facing towards the slide). After a while, the coverslip dries up and the PBS crystal forms.

[bob1]

Ok, you will need to wash off the salts or seal the edges of the coverslip (usually done with clear nail-polish, Revlon make good stuff, don't go for the cheap ones, they don't have enough acrylic to seal well).  If you use a mounting medium such as glycerol, this will slow the drying process too.

[Member2]

bob1, on 16 November 2012 - 07:42 PM, said:

Ok, you will need to wash off the salts or seal the edges of the coverslip (usually done with clear nail-polish, Revlon make good stuff, don't go for the cheap ones, they don't have enough acrylic to seal well).  If you use a mounting medium such as glycerol, this will slow the drying process too.

Ok thanks for the help. I will definitely try that. If I have to wash the cells, then should I just wash with ultrapure water? Thanks again for the help.

[bob1]

If they are fixed then a rinse in water should be OK, especially if you are using the slides immediately.  If you need to keep the slides for a while then you need to use a mounting medium.

[Member2]

bob1, on 19 November 2012 - 12:01 AM, said:

If they are fixed then a rinse in water should be OK, especially if you are using the slides immediately.  If you need to keep the slides for a while then you need to use a mounting medium.

OK thanks, I will try that. Thanks a lot for your help and time.

[Member2]

bob1, on 19 November 2012 - 12:01 AM, said:

If they are fixed then a rinse in water should be OK, especially if you are using the slides immediately.  If you need to keep the slides for a while then you need to use a mounting medium.

When I am preparing p-formaldehyde solution, it turns clear onto its own at < 70 degrees. Do I still need to add NaOH?