Paraformaldehyde is a polymer of formaldehyde. Paraformaldehyde itself is not a fixing agent, and needs to be broken down into its basic building block formaldehyde. This can be done by heating or basic conditions until it becomes solubilized. Once that occurs, essentially they are exactly the same.

Beware though, some commerical formaldehyde solutions contain methanol to prevent polymerization (into paraformaldehyde), and this methanol can potentially inhibit your experiment.

We allow paraformaldehyde to heat over-night, filter, and use fresh for our fixation protocols for immunofluorescence, and we have great success. We store the paraformaldehyde in the fridge, but do not use it after a few days because it will eventually polymerize again and become less efficacious.

Hopefully this helps,

~Adam

Paraformaldehyde is a polymer of formaldehyde. Paraformaldehyde itself is not a fixing agent, and needs to be broken down into its basic building block formaldehyde. This can be done by heating or basic conditions until it becomes solubilized. Once that occurs, essentially they are exactly the same.

Beware though, some commerical formaldehyde solutions contain methanol to prevent polymerization (into paraformaldehyde), and this methanol can potentially inhibit your experiment.

We allow paraformaldehyde to heat over-night, filter, and use fresh for our fixation protocols for immunofluorescence, and we have great success. We store the paraformaldehyde in the fridge, but do not use it after a few days because it will eventually polymerize again and become less efficacious.

Hopefully this helps,

~Adam

Great answers. Excellent. On my last publication you can see the different fixation patter of the fixative when used in flow cytometry applications. To get the best staining you one has to get the right time window. 

All the best

André 

All good answers so far...

I would add that you can "crack" PFA more quickly (using a trick from Milne, Zhao and Hess, 2009):

"10% Paraformaldehyde stock: Microwave 18 mL of PBS in a 50-mL tube placed in a beaker with water until water starts to boil (leave lid of tube slightly ajar). Do the subsequent steps in a hood. Add 2 g of paraformaldehyde (wear a mask when weighing out) and 140 μL of 1 M KOH. Vortex carefully until the paraformaldehyde dissolves. Add additional PBS to 20 mL (about 500 μL). Cool to 37°C before use. Can be stored as frozen aliquots indefinitely. "  Then filter it.

If you have trouble fully dissolving it, your PFA is degraded and you should get some fresh stuff and store it cold as mentioned above.

We always use formaldehyde freshly "cracked" from PFA, as its formaldehyde's stability in solution makes it highly variable in performance over time.

Dear Alexander, while I agree with your statement that "all good answers so far", I would like to comment about your hint on the "trick by Milne, Zhao and Hess,2009"

While it is known for a long time at least by researchers dealing with electron microscopy how to make >[(*) see below] in my honest opinion (IMHO) this your hint to an article is the first reference I ever have seen on how to make (PBS-buffered) FA-solution from paraformaldehyde, "cracked" by microwave treatment. I greatly should appreciate your mentioning the complete reference of (i. e. also title and Journal) to be able to follow their recipe or evaluate what they have done really.

As a benchworker myself (dealing a lot of times with making fresh FA-(and FA-GA) fixatives for electron microscopy) I would like to advise everyone/anyone trying to use such a MW (microwave-) treatment to be as careful as possible, since no parameters necessary or usually to be known when microwaving ( type of microwave apparatus, energy used [Watts] and usually indicated in seconds, eventually use of ? applied how and how much]  have been given here. 18 ml of PBS easily will play < rocket >in your microwave oven if you don't have the right parameters at hand. You microwave (after that reference Milne, Zhao and Hess,2009) just to get the PBS-solution to boiling, then adding the PFA-powder (2g) + 140µl of KOH (= basic component).

*) Compared to that latter approach the "old" recipes for making FA from PFA powder tell us:

Fill in a (250 ml) beaker glass approx. 85-90 ml A. bidest.  Add 4 g. paraformaldehyde powder (use PSA! and use fume cupboard). The PFA-powder will not dissolve by only swirling the tube (at RT). Heat/warm up solution while stirring (hotplate+magnetic stirrer) until 70°C at the maximum, and add -if PFA does not dissolve properly after some minute of stirring,  1 (after the other) to maximally 3 or 4 drops (e. g. from a 2.5 ml plastic one way pipette) of ~2.52% - 4% =~0.55N / 1.0 N NaOH. The solution will clear some second after the right amount of NaOH-alkalinization. The (unbuffered) fixative solution should get as much NaOH(or if you like, KOH) as to give a pH of around 7.0 in the end. 

Let cool down the solution to RT (or, most rapidly, shake the vial under flowing cold tap wate)r and then fill up with A.(bi)dest to end volume of 100 ml. 

The resulting FRESH, monomeric 4% hydrous PFA can be

i) either be aliquoted and frozen for further use

ii) mixed with the appropriate buffer solution for immediate use [NB: if you need 4% FA-concentration in your fixative, start with 8g PFA-powder in approx. 8-85 ml A.dest).

This way you need not making every time fresh PFA solution (if you aliquote PFA-dissolved in buffer you will have polymerization of FA again over time).

Best wishes and regards, Wolfgang

That is interesting that un-buffered FA can be stored reliably...

The microwave-- a common kitchen-type with a rotating carosel-- is simply for heating the PBS to a gentle boil, the PFA never experiences microwaving.

The full reference:

Dear Alexander,

thank you for  trying to add here the full reference (which obviously was not transmitted properly....)

I always try to give full reference data when replying just to make it easy for anyone to follow the intention of what I said. Naturally one can find references nowadays really easily and so did I...(but to be honest: not all ResearchGate-Participants do have the possibility to search with NO limits or without some limits (e. g. using google fromsome countries).

So for all others I include that reference here for convenience :

Thomas A. Milne, Keji Zhao, and Jay L. Hess: in Methods Mol Biol. 2009; 538: 409–423.

doi: 10.1007/978-1-59745-418-6_21 can be found at: 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157307/

(2.2.MB pdf: 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157307/pdf/nihms508315.pdf  )

from there, 

2. Materials

2.1. ChIP with PCR Detection

point  "10% Paraformaldehyde stock: Microwave 18 mL of PBS*) in a 50-mL tube placed in a beaker with water until water starts to boil (leave lid of tube slightly ajar). Do the subsequent steps in a hood. Add 2 g of paraformaldehyde (wear a mask when weighing out) and 140 µL of 1 M KOH. Vortex carefully until the paraformaldehyde dissolves. Add additional PBS to 20 mL (about 500 µL). Cool to 37°C before use. Can be stored as frozen aliquots indefinitely."

*) no definition of pH has been given in the Article/reference.

The last statement of these authors I do not / cannot believe...    because    I F 

FA is dissolved in  a ionic buffer (also in frozen state, which usually means    -(minus)25 °C)  polymerization (and degradation) of FA will take place for sure

( IMHO can only be taken granted for say 1-2 weeks therefore...). 

Yes - and O. K. - I have understood that the Microwave oven only is used for producing HOT / boiling water...  but who cares about the real temperature when dissolving the PFA-powder? 

(FA is obtained by dissolving paraformaldehyde in low concentration sodium hydroxide at 65-70°C. Alternatively PFA is dissolved directly in phosphate buffer  pH 7.6 at about 70°C *)

e. g. cf. 

GLAUERT A.M.: Fixation, dehydration and embedding of biological sepcimens, North Holland Publishing / American Elsevier, 1975

*MILLONIG G, BOSCO M:  Some remarks on paraformaldehyde as fixative. J. Cell Biol,35, 177A, 1967

My chemists senses are tingling here Wolfgang (albeit faintly).  The conditions you provide for cracking PFA to FA are not so different from the ones we are using, and if you suggest that you are preparing 4% FA (little PFA remaining), whereas we are only dissolving PFA at 2.5 higher concentration with little cracking as the reference you cite seems to suggest, I am skeptical.  (By the time we are adding the PFA, the temperature is probably in the 80s or 90s.)  That said, I have no characterization of the formaldehyde/paraformadehyde distribution under our conditions-- do you have such information for yours?

Also, the original reference:

MILLONIG G, BOSCO M: Some remarks on paraformaldehyde as fixative. J. Cell Biol, 35, 177A, 1967.

does not exist on Pubmed, google scholar or the JCB website which has archives spanning back to 1955 (there is no 177A, there is another article spanning that page numbering without the A, and G Millong has never published in JCB, according to their website).  Can you send either a link or the proper reference so I can assess the analytical chemistry that suggests the PFA/FA distribution?  I would be curious to know how much PFA vs FA we have in solution... 

After a little more looking it becomes more interesting.  PFA when freshly dissolved at elevated temperature to drive depolymerization, rapidly hydrates to form methyleneglycol ("MG" which is still electrophillic to amines), which is in equilibrium with the FA aldenhyde form (CH2O), although the more reactive aldehyde is disfavored by three orders of magnitude in dilute solutions.  This is equilibrium reasonably rapid (albeit slower than diffusion of the MG into tissue), and thus most X-linking is a classic Curtin–Hammett driven small population of FA aldehyde rapidly reacting with biopolymers and pulling the equilibrium towards it despite being disgfavoted in the ground state.  

At neutral pH, the distribution between its hydrated form (methyleneglycol) and oligomers of the hydrated form shifts as a function of the (FA WT/unit volume ).  More concentrated solutions like the 10 and 4% (w/v) solutions we are making, have a good deal of methylene glycol dimer (ca ~30, 15% respectively), and to a lesser extent, trimer (technically these higher order oligomers are vestiges of the PFA polymer).  As a function of concentration, these oligomers form over time.

This is important for fixation, as the oliogomers are less reactive is ~in X-linking, and the re-equilibration upon dilution is actually remarkably slow (can be accelerated with pH and temp).  The reading that this thread inspired will definitely make us change our practices. A 4% solution has way more MG monomer and less dimer/oligomer, and is a much better stock solution to make fresh and use as it is more reactive out of the box and does not require a slow MG formation from the oligomers in solution.  Alternatively, one can dilute the 10% "MG" into the cross-linking/fixing buffer and let it equilibrate for an hour or so at the lower concentration (provided that there are no nucleophiles in the buffer, like Tris, thiols, etc.).  The re-polymerization of 40% commercial aqueous FA solutions as they age and slow depolymerization upon dilution from much higher oligoMG probably explains the value of cracking PFA fresh (the heat and pH will form a good deal of MG monomer) more than formate accumulation vis-a-vis cross-linking effectiveness.

This paper has a nice table:

Proton and carbon-13 nuclear magnetic resonance spectrometry of formaldehyde in water.

D. J. Le Botlan, B. G. Mechin, G. J. Martin

Anal. Chem., 1983, 55 (3), pp 587–591

DOI: 10.1021/ac00254a041

Is PFA known to cause more autofluorescence than formaldehyde? 

Dear Alexander,

apologize for my late reply. It seems that I didn't get an info-e-mail by RG that you responded to my posting (as I see now two replies....). My being silent unintentionelly forced also by vacation and otherwise hard work in the lab.

 I would like to answer very briefly here that i) I shall go through your posts meticulously and, ii) naturally I will hunt after the original reference of MILLONIG G and BOSCO M, which seems (at least for now) an old fake self citation by the author Millonig himself ( as I will prove by a pdf copy later on).

For now: thank you for the interesting elaborate representation of the chemistry of FA & MG....I'll be back as soon as possible, best regards, Wolfgang

Edit by WM 2016-11-17,12:05: From answer in another specific RG-thread: "Visualising degree of PFA-fixation in paraffin sections"

(cf.https://www.researchgate.net/post/Visualising_degree_of_PFA-fixation_in_paraffin_sections ) I became aware of the fact that I have been downvoted anonymously.  Despite that and without a chance to discuss a possible cause for that I shall leave this my post [ it was only 26 days before my leaving the EM-Lab due to my retirement without having a successor in that professional function) ....I have answered a lot of similar questions with a lot of upvotes - which are thankfully accepted - one can find easily pasting into RG-URL the following:

https://www.researchgate.net/search.Search.html?type=question&query=Formalin+Formaldehyde    

(apologize for lengthiness)

Dear Alexander:

as promised I would like to present results of my reference/literature search for the unpublished(?) “article” by Millonig and Bosco,1967 (as stated in my post [Reply #07] and critically mentioned in your reply #08 of the thread).

First of all, please find atttached the recipe-page(s) out of Millonig’s , MILLONIG G. (ed) Mario Saviolo Editore/ Publ.Incorp., Casella postale 182, I-13100 VERCELLI, ITALIA © 1976 

as well as the corresponding (self-citation) reference-data for

self citation in:

MILLONIG G. (Ed): Laboratory Manual of Biological Electron Microscopy, Mario Saviolo Editore/ Publ.Incorp., Casella postale 182, I-13100 VERCELLI, ITALIA © 1976  = 

Millonig, G., and M. Bosco. 1967. Some remarks on paraformaldehyde as fixative. J. Cell Biol. 35:177a.

You are right in saying that in the whole archive catalogue of the one cannot find either an

author MILLONIG G (for 1967 cf. http://jcb.rupress.org/content/by/year/1967  or any other time since 1955 )

or the cited title by Millonig and Bosco [ as you said: ].

Nevertheless I found the following interesting (?, perhaps “nice” or better "intriguing") facts:

i)  Courtens JL in his article "The appearance of new electrical properties in the plasma membrane of ram spermatids", published in:Annales de biologie animale, biochimie, biophysique, 1979, 19 (3A), pp.689-693. < hal-00897488 > =  go to:  https://hal.archives-ouvertes.fr/hal-00897488/document

cites explicitely in its text  which can found in the reference list (p.693) as:

MILLONIG G., BOSCO M., 1967. Some remarks on paraformaldehyde as fixative. J. Cell. Biol., 35,177

 ii) cf.: http://jem.rupress.org/content/137/3/776.full.pdf

A STUDY OF ALLOGRAFT KIDNEY REJECTION OCCURRING SIMULTANEOUSLY IN WHOLE ORGAN AND EAR CHAMBER GRAFTS IN THE RABBIT

by JB HOBBS  and   WJ CLIFF, in:

The Journal of Experimental Medicine 137, 1973, 776 – 798, published March 1, 1973

cites as Reference 10:

10. Millonig, G., and M. Bosco. 1967. Some remarks on paraformaldehyde as fixative. J. Cell Biol. 35:177a. (Abstr.)

iii) Last but not least it is amazing that accessing: 

http://jcb.rupress.org/content/39/2/339.full.pdf+html?sid=b251c35f-b430-4567-b250-96dff903216a =

The Fine Structure of pronuclear development  and fusion in the sea urchin, Arbacia punctulata,   by Frank J. LONGO and Everett ANDERSON, 

The Journal of CELL BIOLOGY " VOLUME 89, 1968,   339-368     

one can find the following reference citation:

If staining against/for tubulins, FA has been argued as a more desirable fixative than MetOH due to MetOH's greater potential to disrupt polytubulin structures.

Dear Ulul, just read the specifications on the bottle label and then you will know about the consistence of your fixative (i. e. regarding additives for stabilization like approx. 10% methanol and for buffering the generated acidic cleaving products Ca-carbonate). ...

Edited 2018-03-15,00:40 a.m.:

Vide esp. answers 1 and 2, which have been highly recommended for their comprehensive content of general information

@all (only if interested),.

Regarding my answer #007, the reply #008 of Alexander Ruthenburg and my answers #011 and #012:

With reference to the "G. Millonig" secret (anticipated "wrong reference", "no publication" at all, etc.) I would like to give notice that all I said previously about the mentioned authorship of MILLONIG, as well as MILLONIG&BOSCO, 1967 were and are correct. I have found and saved now the text of the 2 Abstracts, the text of which I shall upload into this thread today in the later morning. Regards W.M.

EDITED 2018-04-26...it is right early this morning... gladly I dound this thread again ....it seems nobody took notice meanwhile... Nevertheless: apologies for my forgetting to upload the file I promised.... (layout may not be be perfect....but - at least -readable AND - most importantly: G. Millonig and Bosco rehabilitated... :

Further reading:

KIERNAN John: 'FAQ's in Histology'

"there is no paraformaldehyde-solution" but a "formaldehyde solution made freshly from (with the use of) para-formaldehyde powder":

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

search for either "Formaldehyde" OR " Paraformaldehyde" and

(CTRL F) find:

** Paraformaldehyde: why won't it dissolve? (Answer includes other information about formaldehyde and fixation)

Nice article by D.J. Le Botlan et al , which was shared by Alexander .

and thanks RG which is excellent platform to learn lot of information.

Thanks Adam J Case for your answer. It saved my day.

I´m quite confused with this subject. If I have a commercial 4% paraformaldeyde solution, as it is already soluble, that is equivalent to a 4% formaldehyde solution?

Article Formaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde:...

Adam J Case What about commercially available glass vial 32% PFA solutions (https://www.thomassci.com/Chemicals/Solutions-F/_/Paraformaldehyde-Formaldehyde ). I have just been diluting to 4%, do I need to be heating as well?

Mandy Herring , [The following sentence deleted 12 minutes after initial Posting due to "offensive content" as of Mandy Herring's reply below)

--------------

Original content:

[I start my answer here a second time due to loosing all I had written before by just clearing up the profile URL of Prof. Dr.John A Kiernan (cf: John A Kiernan )].

Unfortunately, the US COMPANY Thomas Scientific Incorporated = Thomassci.com [cf.: https://www.thomassci.com/]

still claims (at least for me in SALZBURG, AUSTRIA, EUROPE)

cf.: https://www.thomassci.com/EUGDPRStatement.htm : (qoute as of some seconds ago): " We're sorry, due to the EU GDPR requirements we are unable to service content at this time. We are working hard at making our website compliant as quickly as possible. Thank you for your patience and understanding.

Please check back soon." end of quote;

NB: the completely blocked pages appear also blocked when trying to use the information in the CACHE files) so I cannot help you with an URL of their MSDS (Material &Safety Data Sheet) or TDS (Technical Data Sheet) for their (for sure) hazardous product "32% Paraformaldehdye solution in glass vial". Usually such an item is packed under either inert gas or "sans air" and sealed properly (heat-sealed glass ampoules), shipped and stored usually at decreased temps (cooled at least to 4°C or "on dry ice"). Further storage either at 4°C (some also freeze, but that depends on the available empty space + in vials for the possible expansion when freezing and should be included a note in the safety instructions). So you should have received or at hand at your work place MSDS or STD enclosed / together on delivery of the ampoules.

Regarding your question: '..., do I need to be heating as well?' the answer is short: NO, you need not, unless you - for another reason - want to use 'warm' fixative.

The former PFA-powder already has been solved in a correct fashion to yield the / a highly pure 32% FA-(=formaldehyde) solution.

The "brand" name /denomination: "PFA" ( as you wrote: 'glass vial 32% PFA solutions' ) IMHO just only wants to point to the fact that the solution has been produced from PFA-powder as initial component.... -

instead of using Formalin[(R) or (TM) of MERCK] which usually is sold as hydrous solution up to max. 38% concentration (e.g. for Use in Histology] BUT stabilized with approx. 10% MetOH and Ca-Carbonate, & therefore free of formic acid.... or other initial sources (therefore: see / cf. MSDS / STD / or last but not least: the Certificate of Analysis)

There exist patents on how high purity PFA-solutions can be made: cf. e.g. ,

'Process for producing high purity formaldehyde', https://patents.google.com/patent/US4962235A/en

Since the concentrated 32% PFA stuff is made up free of impurities (i. e., 'dimers', 'trimers',..... 'polymers'... which might either impede or inhibit fast and thorough penetration AND correct fixing action of the fixative) this is expected to be kept also for storage, during transport and delivery.

The ampoules therefore are sealed rigorously to be and to stay chemically inert (see above) for an unwanted oxidation, usually those ampoules also are shielded from light by shipment in closed boxes usually at ambient temperatures or sometimes also at 4°C/cooled on ice.

Finally: we do not know how (with which solution) you diluted your 32% high concentrated PFA already:

hopefully:

1) (bi-, tri-)destilled water or:

2) a suited buffer solution.

In case 1):

storage after opening a new - originally sealed - ampoule the dilution should be used (as always: also depending on the task) within up to 3 hours at RT. It is recommended to to use all content of the vial to make appropriate solution volume(s). Small volumes might be frozen (e.g. 2 ml in Eppendorfs, finally sealed with parafilm)

Storage at 4°C in a glass vial (or, e.g., an Erlenmeyer conical flask with narrow neck and tight fitting plastic stopper) either overlayed with inertgas or stoppered 'air-free' for most applications (in IHC, Histology, etc. for instance) for up to one or two days or even longer [ if the 'container' isn't opened often!] might be possible.

Delicate experiments perhaps need analytic proof (e.g., spectro-photometry) of being free from polymer formation.

In case 2):

Storage for longer than necessary can not be recommended, but as an own experience might be [premises: storage at 4°C =in the dark), and free from air/oxygen] up to one week (example: if opened twice, e.g. for tissue fixation in diagnostic routine). The duration of time the buffered fixative can be used effectively also may depend on the 'buffer' one uses (e.g. anorganic like phosphate buffer, organic like sodium cacodylate vs. zwitterionic buffer like PIPES, HEPES or something like that, and how 'clean' the making of has been done.... The more basic the fixative / solution, the quicker the / a polymerization will occur.

Hope that is of any value for you, best of luck, WHM

Wolfgang H. Muss What on earth does me being a Broad Institute member have to do with asking questions? Am I supposed to just know everything because of where I work?

If this my reply was offensive to you: I REALLY apologize.

Short explanation: when using the "MENTION FUNCTION" @ like Mandy Herring

automatically the affiliation is displayed too (so everyone using ResGate can see your affiliation)

Affiliation📷Broad Institute of MIT and HarvardLocation

• Cambridge, United States

Department

• Stanley Center for Psychiatric Research

Position

• Research Associate II

If you don't want to read information which has been categorized 'OFFENSIVE' then you are free to neglect reading.

The facts nevertheless still are there and physically proven.

You are also free to report my offensive reply to your department or to ResearchGate.

Best of luck again.

(edited after some seconds of initial posting for minor misspellings and omission of words, and a second time)

Wolfgang H. Muss You still did not answer my question-- WHY does it "amuse you" that a "Broad Institute of MIT and Harvard institutional member need to ask such a question". We ALL have questions, no matter which institution we come from. Your answer didn't need to be offensive, you chose to MAKE it offensive by suggesting that I, as a Broad Institute member, should already have the intelligence to know the answer to that question. I ask and give answers on Researchgate so that OTHERS who have the same questions can also find information. If you thought my question was dumb, fine, but your pompous "amusement" comment did NOTHING to answer my question and was uncalled for.

Does it need - after all your sentences - any comment from my side?

Then why reply :)

Well that escalated quickly!

Mandy Herring To answer your question, I think you should be good without heating as long as you do not see any precipitation form (i.e. paraformaldehyde polymerizing) in the solution. The website states it is methanol free, so that will lower the stability and I am guessing the time in which you can use it effectively. At the end of the day, if you are not seeing any type of precipitate and it is effectively fixing your tissue (depending on your use, you can usually tell) then you should be good to go. If you feel your solution loses its potency overtime or forms a precipitate, put it in a 60 degree C water bath and that may help. Hope that helps!

~Adam

Adam J Case Thanks Adam, that’s all I was looking for :) I have sealed glass ampules of 16% formaldehyde (Thermo) but I was also just given 32% PFA in a plastic container (dark plastic, original bottle of that same EMS brand) to be used for FISH, which I just started learning. I had always made my own PFA from powder or used the concentrated sealed vials of 16% formaldehyde. My probe signal has been low I was concerned about the air exposure of repeatedly opening the plastic 32% PFA container and possibly not fixing properly and I thought perhaps I missed something from the product sheet about a heat treatment before using but couldn’t find anything online. I am doing a parallel experiment tomorrow with the two formaldehyde brands, as well as two different fixation times since I wasn’t sure, so we will see! Much appreciated.

Mandy

i fixed mice liver samples in 4%PFA for 24hrs then cryoprotected them with 30% sucrose and then in OCT and then freezed them in cryostat. but after cutting the samples I'm getting poor morphology of the samples. Can anyone help?

Comprehensive answer by @Adam J case. Both formaldehyde or paraformaldehyde can use for fixation cell. Depend on your protocol

@ AdamJCase , would you or anyone else be able to comment on the appropriate selection of formaldehyde vs paraformaldehyde and their concentrations when two different fluorophores are used in a protocol - one requires paraformaldehyde, one requires formaldehyde? Thanks

Allyson Cochran They are literally the same thing. Paraformaldehyde (powder) in solution becomes formaldehyde. Use 2-4% final concentration in PBS on cells for fixation, 15 minutes at room temperature.

Adam J Case

Thank you so much

Adam, that’s all I was looking for

Allyson Cochran : I would suspect, the one requiring "paraformaledehyde" explicitly, might be the one more sensitive to common additives found in commercial formaldehyde solutions. You should be fine by using only formaldehyde solution from freshly depolimerized paraformaldehyde for the whole protocol.

Cheers, Paavo

Paavo Bergmann and Jordan R. Yaron thanks very much. That's exactly the info I was looking for!