Dear Abdolvahid Sadeghnejad

Formalin is the common name for saturated (37%) formaldehyde solution, therefore 10% formalin is approximately a 4% formaldehyde solution. People fix usually in 0.5-2% PFA (paraformaldehyde) to crosslink proteins and retain their antigens. Paraformaldehyde (PFA) is polymerized formaldehyde, the PFA you use for fixation is "pure", methanol-free formaldehyde made by heating solid PFA and if you wanna be a stickler it's misnamed as it is not in its polymer form.

Please see:

https://microscopy.duke.edu/guides/paraformaldehyde-formaldehyde-formalin

for clarification and

http://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf for the protocol I have followed to prepare PFA for my FACS experiments.

All the best,

Michael

Thank you so much dear Michael Förster

That was really helpful

Exactly the problem is that "Methanol" for my large molecules like PE fluorochrome. So i have to use PFA.

Do you have any idea for making this pure free-methanol PFA from that 12%methanol 37% formaldehyde ? or i force to buy PFA powder and make the solution from your protocol?

Thanks indeed

Abdolvahid

Hello

Formalin is the name for saturated (37%) formaldehyde solution. Beware though, some commercial formaldehyde solutions contain methanol to prevent polymerization (into paraformaldehyde). Since 100% formalin contains up to 15% of methanol as a stabilizer, it has a significant impact on cell fixation.

Methanol is a permeabilizing agent. It can interfere with the staining of membrane bound proteins, and can greatly influence staining of cytoskeletal proteins. For example, when staining cellular F-actin it is imperative to use a methanol-free formaldehyde fixative. This is because methanol can disrupt F-actin during the fixation process and prevent the binding of phalloidin conjugates.

"Pure" methanol-free formaldehyde can be made by heating the solid PFA. 4% paraformaldehyde is usually made in PBS or TBS at 70 °C with several drops of 5N NaOH to help clarify the solution. Prepare 4% paraformaldehyde solution in a chemical hood and then store in a refrigerator. Because the solution will re-polymerize during storage it is best to use immediately or within a few days.

Reference:

https://www.aatbio.com

Good Luck

Hello Saleh Alkarim

Thanks for your answer

In addition, I found more details about explanation of biological surface or intracellular proteins, different fluorochromes and kind of fixatives.

This is the link:

https://bitesizebio.com/22141/fixation-and-flow-cytometry/

For nomenclature:

http://www.cyto.purdue.edu/cdroms/cyto3/1/topics/prep/0011.htm

Hope you the best