The cloudy suspension is most probably caused by formaldehyde polymers. They are broken down while heated. Not using any buffering agent in your solution is a bigger problem. For the future you should use PBS.

In my lab we are dissolving PFA powder in water with few drops of NaOH, heating it and when it is dissolved adding 10X PBS to have a final concentration of 4%PFA 1xPBS, then we adjust the pH.

Keeping homemade solutions at 4 degrees for a prolongued time is not a very good idea. Formaldehyde molecules can react with each other to form methanol and formic acid. 

If you want to read more about the biochemistry of formaldehyde, paraformaldehyde and its water solution, check:

http://publish.uwo.ca/~jkiernan/FixAnti2.pdf

http://publish.uwo.ca/~jkiernan/formglut.htm

Good luck with your experiments!

Hi Andrea,

In our lab we use a 1% and 4% PFA solution and we dissolve it in PBS buffer (for perfusion or incubating brain sections). I can't speak for an 8% solution but the 4% solution is heated in a water bath with a magnetic stirrer until it's completely dissolved. This takes at least a couple of hours, sometimes overnight (depending on the volume). Afterwards we freeze it -20°C and when it's thawed again completely it doesn't have the cloudy appearance as you describe it. My guess it has to do something with the NaOH you add (thus changing the pH). I would suggest either to change to PBS instead of water (if possible) or use liquid PFA to avoid using NaOH to dissolve the powder. It depends on the application, do you want to perfuse or do you incubate fresh cells/sections?

Good luck,

Jeroen

Thank you Jeroen for your answer. I'm sorry I forgot to say that I adjust the pH of the solution to pH 7.4 just before filtering. Your protocol seems to be the one I used until few months ago (except I used water instead of PBS).  Concerning the application, I incubate fresh cells. My immunostainings seem to be fine now, but I had some variability in the efficacy. I just wanted to understand why I had this "problem" (if the cloudy suspension is actually a problem, I don't know) and possibly find the best way to prepare my solution. I started adding NaOH because I found it in several protocols, apparently PFA dissolves better in a basic environment. Thank you again for your answer!

Andrea

The cloudy suspension is most probably caused by formaldehyde polymers. They are broken down while heated. Not using any buffering agent in your solution is a bigger problem. For the future you should use PBS.

In my lab we are dissolving PFA powder in water with few drops of NaOH, heating it and when it is dissolved adding 10X PBS to have a final concentration of 4%PFA 1xPBS, then we adjust the pH.

Keeping homemade solutions at 4 degrees for a prolongued time is not a very good idea. Formaldehyde molecules can react with each other to form methanol and formic acid. 

If you want to read more about the biochemistry of formaldehyde, paraformaldehyde and its water solution, check:

http://publish.uwo.ca/~jkiernan/FixAnti2.pdf

http://publish.uwo.ca/~jkiernan/formglut.htm

Good luck with your experiments!

If you keep the PFA in 4 degree C the cloudly stuff will settle down. Then just transfer the supernatant to a new container and use it. It works perfect for immunofluorescence. Try to avoid NaOH for dissolving as it increase background staining for certain fluorescent dyes such as Hoechst33342. 4% PFA is just fine. I don't think you need 8%.

have you tried purchasing PFA that is already in solution rather than dealing with dissolving the powder?  PFA in powder form is nasty to make up and we purchase PFA in solution from EMS.  We use 4% made in PBS++ and it works great for our confocal microscopy experiments.  I do not believe that you need to use 8%.  That seems a bit too high to me.

http://www.emsdiasum.com/microscopy/products/chemicals/formaldehyde.aspx#15700

Thank you to all of you for your answers, and thank you Agnieszka for the precise details, they're really helpful. I cannot make a solution less than 8% because the protocol I use for my particular cells requires a 6.5% PFA fixation step, so I'm obliged to start from a stock solution higher than 4% in concentration. But the fact that I dissolve it in water without adding any buffer might indeed explain the bizarre behavior I often observed. Thank you again!

Dear Andrea, just to ask one detail I haven't :

in your last Re you say you / your protocol need(s)/require(s) for the fixation step.

So what is the logics to make a stock solution of 8% fresh PFA from the powder? Why you don't set up 6.5 % PFA solution? Is it because you have to mix then with PBS, or any other buffer solution?

If yes, which buffer? (usually pH of such solutions will be adjusted via adding [PO4 = phosphate] buffer solution 0.05, 0.1 or even 0.13M, if the buffer vehicle shall be isotonic with hum. blood.

Also it would be important to know something about the specimens you are fixing then: single cells (e.g. on cover slips), cultured cells?, tissues (tissue blocks)?

By the way, all you want is to do, Agnieszka Piszczek  has said valuable facts about your  (do you want to know how to prevent this? and want to know what this eventually means in terms of fixation quality? just reply and ask, because I would not like to bother any of the posters so far).

From some answer seen I feel that only little is known about making fixatives, especially PFA solutions chemically correct.  There are a lot of threads in RG concerning this "problem" some of which I could recommend to read.

Best regards,

Wolfgang

Formaldehyde polymers probably cause the cloudy suspension.

They can’t become clear by heating only. The only way to make the solution clear by adding a few drops of NAOH. Then adjust the PH with high concentration of HCL up to 7-7.4 and add PBS.

The best way is to prepare the solution freshly each time. This solution can also be preserved for one month at –20 C not 4C

Good luck,

Dear Meiling Chen, I do not agree with your statement:

"Then adjust the PH with high concentration of HCL up to 7-7.4 and add PBS"

Why:  because it is certainly wrong.

As you said correctly: "adding a few drops" (NB: what is:  a few drops?)  of NaOH (basic) you'll get at least a solution of paraformaldehyde solution near neutral pH. Adding HCl (acidic) would decrease the pH (especially if using, as you state: !"high concentration"!).

As a standard in fixing e.g. also TEM-specimen preparations the following procedure is easy:

for 8% PFA-solution (unbuffered) add (cautiously, don't breathe in, use personal safety utensils (goggles& gloves)

8 g PFA-powder into a beaker glass [100-150 ml volume]  filled with ~80 ml A. bidest. (or better quality if needed).

Heat on a heating device (best: magnetic stirrer with hotplate) and control temperature all the time until 60-65 degr.C are achieved. Don't heat over 70°C.

Then, under constant stirring:  add drop by drop (e.g. from a one-way plastic pipette 2.5 ml) 1N NaOH - waiting after every drop for a change in physical presentation of the milky solution:  When the solution becomes clear, don't add more NaOH. 

Let cool down the hot solution to room temperature, and fill up to 100 ml End Volume (use calibrated/graduated glass cylinder or Erlenmayer flask). Eventually filter the solution through Whatman #1  filter paper (or similar product). Storage in tightly stoppered 50 or 100 ml Erlenmayer flask at 4°C at the maximum for 3-4 days, freezing to -20 °C (or better  -80°C) could prolong the time the solution will be free of di-, tri,...polymers of FA which will interfere for sure with fixation efficiacy.

NB: not to forget formation of formic acid and other degradation byproducts over time.

Then use the 8% PFA solution to dilute it with buffer (Tonicity xxx, pH yyy)  as appropriate.

CH Fox, F B Johnson, J Whiting and P P Roller: Formaldehyde fixation. In: J .Histochem Cytochem 1985 33: 845ff,  DOI: 10.1177/33.8.3894502 cf:  http://jhc.sagepub.com/content/33/8/845.citation

see also: Fixation Strategies and Formulations used in IHC staining,  http://www.piercenet.com/browse.cfm?fldID=B7273393-CD71-46C0-A8A7-E702C294529E

 Thank you for your answers. Dear Meiling, actually the cloudy suspension disappears after mild heating and vortexing.

Dear Wolfgang, your second answer describes exactly the protocol I use, except that, after having completely dissolved the powder, I adjust the pH to 7.4 with HCl. Anyway, if I go back to -20, I will get the cloudy suspension again (probably because of the lack of a buffer solution like PBS as Agnieszka suggested?) I will read the articles you kindly gave me, thank you. 

Best, 

Andrea

Dear Wolfgang. I am wondering what is the benefit of keeping the frozen solution of PFA powder unbuffered and diluting it in PBS afterwards?  Formaldehyde binding to the tissue is faster at pH close to neutral than acidic, so I assume it would be beneficial to have some buffering agent in the solution which is brought to the apropriate pH to counteract the acidification. But that is just my feeling and I would be interested if there are any reasons against this approach. 

We usually dissolve PFA in 1xPBS at 65 degree centigrade to afinal concentration of 4%  without adding NaOH. This solution could be stored stably at 4 degree for up to two weeks. This is our experience. 

I always use only freshly made PFA, epecially for EM fixations. I also heat distilled water to dissolve the PFA and add NaOH. To my mind, it is the amount of  NaOH that is essential. If you use 1g PFA in 12,5 ml of water 1-2 drops of NaOH is enough. Then when the solution is cooled to room t, I dilute it with buffer. Storing PFA leads to formation of formic acid, and it is not recommended at least for EM fixations. 

Apologize for lengthiness

Today, on 2nd of July 2016 I found out - due to the most recent Upvote of my Re# 09 by (thank you!) that I promised  Andrea to "come back later on......"    -  see below  -  but lost sight of this thread due to other activities regarding my professional work and  last but not least, also on RG. Today I also got aware that my Reply # 11 (which I delete now but include here following just for information) was downvoted twice...Unfortunately a DOWNVOTE does not initiate an automatic RG-information to the responder, so I was not aware of my fault not to comment on the "clouding phenomenon" as originally intended.

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Original Reply no. 011 (deleted 2nd of July 2016):  

Wolfgang H. Muss · 157.36 · 149.79 · Paracelsus Medical University Salzburg

Dear Andrea, you're welcome.

I shall come back later on to elucidate the phenomenon. Just have to get back to specimen preparation....

Best wishes & Regards, Wolfgang

1 / 2 · Feb 5, 2015

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So I want to reply multiply as follows:

@ those mixing PFA (solution) or dissolving PFA-Powder in "PBS" (e.g. also to Meiling Chen):  I cannot say anything about a correction of the pH with "high concentration of HCl" when not knowing which PBS was used.... I am aware of many erroneous statements made for "using PBS"  instead of "using PO4-buffer", the latter being usually self made knowing exactly about ingredients, molarity as well as tonicity. "Fixative(s) for  morphological examination as well as histology, and immuno-methods usually are prepared using "buffer solutions" which maintain the pH at the level / value  they have been mixed together (cf. diverse handbooks and web-sources on e.g.:  "how to make a [ phosphate ] buffer solution pH 7.4" (for Google:  approx. 955,000 results)).

@Andrea Frapporti: reevaluating your question as well as your answers I found your statement in Re # 006: "But the fact that I dissolve it in water without adding any buffer might indeed explain the bizarre behavior I often observed",  so I can assume that the cloudy precipitate appears after the following process:

  i) dissolving the PFA-powder in "water" (simply guessing it is at least A. bidest ! not only deionized water! ))

ii) as of your information in your original question: "Now I switched to a more precise protocol were I add few drops of NaOH to the solution while stirring it to help dissolve the powder (always being sure not to go beyond 65°C), I filter and I freeze. Now, with this second protocol, my PFA solution presents a cloudy suspension immediately after thawing. I can re-dissolve it by vortexing and warming it up a bit (like, 30°C)....

"Compound hydrous solutions of chemicals" after freezing  might not have the same / identical physical properties after short thawing. Separated phases might be expected unless some action (like shaking/vortexing and some kind of warming) are applied.

The cloudiness of your "suspension" could be also due to condensation of impurities in water (if you used e.g. deionized water instead of bidistilled one), but as well as due to formation of hydroxide precipitation (since we do not know HOW MUCH OF WHICH CONCENTRATION you had added prior to freezing.

Note: there might be a huge difference in correct "alkalinization" of the PFA-POWDER in solution if you use   "3 drops of 0.1 N, OR 1 N,   OR 2.5 N OR eventually 5N NaOH",   Depending on the latter it might be that the pH of the pure PFA-solution (without any buffer solution added) is so alkaline / basic that you need "high concentration of HCl to compensate and titrate back to a "physiological" pH of say 7.4.  

I don't know why (if I got that right) you adjust the PFA-solution proper to a "pyhsiological pH of 7.4"  and then eventually dilute again with "buffer solution " or "PBS" pH7.4.

IMHO the "(P)FA in solution" which has been depolymerized by i) heating to ~65°C and ii) adding some amount of  (0.1N, or 0.5N, or 1 N NaOH-solution) must not be adjusted to a physiological pH at 7.4 because usually in the second step you will add the phosphate buffer solution ("buffering at  pH 7.4")  only to end up e.g. with 1% or 2% or 4% or 6.5% or 8% FA (made from PFA-powder) IN 0.1M or 0.13M phosphate buffer pH 7.4"  

I admit that it  is difficult to explain more thoroughly how those FIX and BUFFER solutions usually are made IN and AS  stock solutions and mixed together / diluted against one another

@Agnieszka Piszczek:  "Freezing unbuffered, freshly prepared PFA-solution"

I assume that at least for one or two days cooling and for some days more freezing CAN prolong the properties of polymer-free and formic acid free fixative solution. But then (for freezing, best at -80 °C) you must not use either additional buffer solution or use of HCl for adjusting the pH as necessary. 

For me - personally - I can't believe that someone really prepares "12 ml of fixative" (cf. Re no 13 by Elena V Sabaneyeva ) only for practical reasons. Usually, 50 or 100 ml are practical volumes to be made.

 it might be that freezing alters the "molecular" configuration, the dissolved chemical components by dissociation in a way that initiates cloudiness (impurities, hydroxide, or even polymeric FA again...   which dissolves completely only on vortexing = bringing into solution again and warming again /heating ).

I am sure if you follow the preparation of PFA-fixative as reported in literature e.g. from John Kiernan that you won't have a problem again.

Discussions and articles regarding the preparation of formaldehyde solution (strictly methylene glycol) by dissolving PFA in water or buffer almost invariably assume that depolymerisation is more or less immediate, based on the formation of a clear solution. I have yet to read an article that actually demonstrates this to be the case (e.g. following the progress of depolymerisation with 13C-labelled paraformaldehyde by NMR) and I would be grateful to anybody who could direct me to one. I was led to understand that depolymerisation took some time, and that initial preparations of PFA in water should be stored over marble chips (to remove any formic acid) until such time as required. Thoughts?

Hello, dear Christopher, may I just jump in to comment to your really interesting task:

For now I don't have any specific (chemical) research article at hand....

Regarding your quest: " I was led to understand that depolymerisation took some time, and that initial preparations of PFA in water should be stored over marble chips (to remove any formic acid) until such time as required. Thoughts? " I would like to add that my understanding for the white powder one could find in former days (I guess until the 1990ies) at the bottom of FA-bottles (esp. from MERCK) was "natural dolomite powder" (lately changed then to "Ca-Carbonate powder"

cf.:

https://us.vwr.com/store/product?keyword=Formaldehyde%20Calcium%20carbonate

"Learn more about Formaldehyde ≥37% in aqueous solution stabilized for histology free from acid and Calcium carbonate. "

==>

Formaldehyde ≥37% in aqueous solution stabilized for histology free from acid and Calcium carbonate Supplier:  MilliporeSigma Description:   Min. 37% Free From Acid Stabilized with about 10% Methanol and Calcium Carbonate*) for Histology *) which is a bit misleading, since stabilisation is brought up by the addition of Methanol (usually - for histological use 10-20%).

Ca-Carbonate (or in former times, natural dolomite powder ) is added to adsorb formic acid which is produced on longer storage of the solution due to permanent 'deteriorating' and polymerizing process. If the carbonate powder is in the bottle you will not be able to discriminate what is Ca-carbonate powder (in excess!) and what may be perhaps polymerized "para-formaldehyde [powder] as it is formed on storage , e.g. at low(er) temperature(s):, cf..

https://us.vwr.com/store/catalog/product.jsp?product_id=4545763:

"Stabilization: Stabilized with methanol 10-15 %

Clear, colorless solution. Caution: Trioxymethylene precipitate can be formed at ppt levels upon standing below 15°C (59°F). Nonhazardous polymerization may occur at low temperatures, forming paraformaldehyde, a white solid."

So - in the end - if you have (if you mill that you should get the "natural dolomite powder") at hands, you could use that for keeping your FA-solution free from (formic) acid...

(cf.: Dolomite ≥85%, granulated < 20 Mesh Supplier:  Spectrum Chemicals Description:   DOLOMITE, MINIMUM 20 MESH, GRANULAR, an aNHydrous carbonate mineral, can be used as a pH buffer in potting soils and saltwater aquariums........

https://us.vwr.com/store/product?keyword=Formaldehyde%20Calcium%20carbonate%20dolomite or:

https://us.vwr.com/store/product/21572834/dolomite-85-granulated-60-20-mesh

Quotes and company names have been taken from sources just to point to the basic data but doesn't mean I am affiliated or have any financial interest in mentioning a certain company name or their product here. Thank you.

Best regards and wishes and good luck...if you don't mind I greatly should acknowledge to read about the outcome of your recent study/studies.