I need to detect and measure the abundance of cytoplasmatic proteins (AIM2, IFI16, GBP1, GBP4) using direct detection by flow cytometry with fluorochrome-conjugated antibodies in human mo-DCs and I need to decide the best fixator to my assays. Someone have some advice and experiences? Formaldehyde or paraformaldehyde?
Dear Amanda,
Formaldehyde fixatives are made by diluting a commercially available stock 37% solution. The solution has methanol added for stabilization. Paraformaldehyde fixatives, in contrast, are made by dissolving the solid form of paraformaldehyde (powder or prill) by carefully heating it in water in order to break the chemical bonds of the ring of the paraformaldehyde molecule to create the formaldehyde solution. This solution is cooled, and then combined with the buffer of choice. Paraformaldehyde -based fixatives have a much shorter shelf life, even at 4 C, since there are no added ingredients that stop the chemical degradation of the formaldehyde. Many of us who use paraformaldehyde based fixatives prepare them shortly before use.
Each type of fixative has its drawbacks. I prefer paraformaldehyde so that I avoid the methanol and I find I can easily control the pH and buffer salts. But I used to perform electron microscopy, so I am familiar with its preparation. Preparation of neutral-buffered formalin is also straightforward, however, and can be much more convenient.
As to which will work for your antibody labeling, check the protocols provided by the company or the scientific literature.
Good Luck!
Jill
Formaldehyde (FA) = gas
Paraformaldehyde (PFA) = powder,
so the difference is (obviously) only the (physical, aggrgate) state of matter (this said only with regard to the textual formulation of your request).
If you want to get answers regarding the effect of the use of hydrous solutions of FA and/or PFA or FA- vs(=versus) PFA-FIXATION-solutions then - please -
visit all those reliable answers in RG-thread one already can find using the full-text Archive-Search (see this webpage, middle of the upper menu-line "SEARCH for Researchers, publications and more..." insert your keyword(s):
" FA Formaldehyde PFA paraformaldehyde fixation " and or " Effects of fixatives fixation on intracellular staining " and so on...
(inserting keywords into the request mask / Search for rectangle, eg.:
| FA Formaldehyde PFA paraformaldehyde fixation |
will create the following URL: https://www.researchgate.net/search.Search.html?type=publication&query=FA%20Formaldehyde%20PFA%20paraformaldehyde%20fixation ),
then choose the results from the respective section , as there are:
- researchers
- projects
- publications or
- questions
- jobs
- institutions
- departments
or (proposal 2):
insert Keyword(s):
| FA Formaldehyde PFA paraformaldehyde fixation flow cytometry | ==> https://www.researchgate.net/search.Search.html?type=question&query=FA%20Formaldehyde%20PFA%20paraformaldehyde%20fixation%20flow%20cytometry and choose from submenue as pointed to above.
Finally, to arrive at least for a very short answer of your question:
As long as you don't use too high FA- or PFA-concentration, choose the right FA- or PFA solution (i. e. perhaps methanol-free for the former, and -for the latter - prepare the PFA-solution physically AND chemically correct ), don't apply /or treat your cells TOO LONG with your - ideally isotonic - hence correctly buffered solution (=buffered fixative) I guess there isn't much difference in intracellular staining of cells comparing FA- or PFA-solution(s). Your success regarding intracellular staining after using flow cytometry rather might depend on the proteins you want to detect/localize and/or the antibodies you used with your technique....
Not to forget: cf. similar (one= same matter) questions: cf:
https://www.researchgate.net/post/How_can_I_decide_the_best_fixator_agent_to_my_cells_for_intracellular_staining_using_flow_cytometry
(reply already by Abdolvahid Sadeghnejad) and, e.g.
https://www.researchgate.net/post/Commercially_available_PFA-what_to_chose_for_cell_culture_fixation
Q by: Abdolvahid Sadeghnejad
https://www.researchgate.net/post/Can_i_use_formalin_instead_of_paraformaldehyde_for_fixing_in_flow_cytometry
And yes: there have been quite a lot of questions dealing with FA & PFA fixative(s) and their proper use - especially their use in properly buffered fix-solutions....
Best wishes and good luck....
Dear Amanda,
Formaldehyde fixatives are made by diluting a commercially available stock 37% solution. The solution has methanol added for stabilization. Paraformaldehyde fixatives, in contrast, are made by dissolving the solid form of paraformaldehyde (powder or prill) by carefully heating it in water in order to break the chemical bonds of the ring of the paraformaldehyde molecule to create the formaldehyde solution. This solution is cooled, and then combined with the buffer of choice. Paraformaldehyde -based fixatives have a much shorter shelf life, even at 4 C, since there are no added ingredients that stop the chemical degradation of the formaldehyde. Many of us who use paraformaldehyde based fixatives prepare them shortly before use.
Each type of fixative has its drawbacks. I prefer paraformaldehyde so that I avoid the methanol and I find I can easily control the pH and buffer salts. But I used to perform electron microscopy, so I am familiar with its preparation. Preparation of neutral-buffered formalin is also straightforward, however, and can be much more convenient.
As to which will work for your antibody labeling, check the protocols provided by the company or the scientific literature.
Good Luck!
Jill
Dear Amanda
Formaldehyde is similar to paraformaldhyde. The difference is that paraformaldehyde is polymerised product of formaldehyde and needs heating to convert it back to formaldehyde. Formaldehyde is a gas dissolved in water/methanol usually 37%v. In histology we use 10% buffered formaldehyde to fix fresh tissue samples. Universally formalin is accepted as a best fixative for most of histological analysis, these include: Immunohistochemistry, In-situ hybridisation, PCR based method etc. From my experience, I would use 10% buffered formalin and this can be commercially purchased.
https://www.sigmaaldrich.com/catalog/product/sigma/ht501128?lang=en®ion=GB and this can keep well for months. Either paraformaldehyde or formaldehyde you will need to figure out antigen retrieval method. This step will unmask antigens caused by formalin fixation. However, as Jill already mention you need to check your protocol and manufacturers recommendation. Hope this helps.
Best wishes
Alex
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