Low concentration (e.g. 2% to 5%) of formalin solution is widely used for well-established formalin pain test in rodents. But can I use the same as the formalin solution for fixative (such as the 4% PFA in PBS buffer)? How do you prepare it for formalin test? Thanks a lot!
Hi, Jiahao,
We prepare in saline. Here is the recipe:
5% formalin in saline
50 μl formaldehyde solution (37 wt. % in H2O)
950 μl sterile normal saline (0.9% NaCl solution)
Best regards,
Ana
Dear Jiahao Ye, and dear Ana Claudia,
if you are mixing up commercially available concentrated Formalin (= usually approx. and maxim. 37% Formaldehyde in water) you know that this solution also contains about 10% Methanol as stabilizer? O.k., you are using it as pain test in rodents...(;-(), so it would be recommended (if the pain test reagent should be free of any other component/reactant) to mix 2, 4 or 5% FA-solution made fresh from paraformaldehyde powder and to apply either undiluted as hydrous FA-solution or diluting this one then with "physiological saline" .
cf: http://www.ncbi.nlm.nih.gov/pubmed/1454405
Pain. 1992 Oct;51(1):5-17.
The formalin test: an evaluation of the method. (NB: Abstract only without methodical data), by Tjølsen A , Berge OG, Hunskaar S, Rosland JH, Hole K.
(This article can be found too with its abstract only at: http://www.sciencedirect.com/science/article/pii/030439599290003T (ToC of PAIN 51(1),1992:@ http://www.sciencedirect.com/science/journal/03043959/51/1
Comment in: More about the formalin test. [Pain. 1993] ....More about the formalin test, Page 237 by Neoh Choo-Aun (http://www.sciencedirect.com/science/article/pii/0304395993900865, no abstract, no pdf).
The article:
by Daniel Le Bars, Manuela Gozariu, and Samuel W. Cadden in Pharmacological Reviews December 1, 2001 vol. 53 no. 4 597-652 (cf. http://pharmrev.aspetjournals.org/content/53/4/597.full ) gives an overview about the field, stating in
VI. Tests Based on the Use of Long Duration Stimuli (“Tonic Pain”),
A. Intradermal Injections:
>......A 0.5 to 15% solution of formalin injected into the dorsal surface of the rat forepaw provokes a painful behavior...... >, in section
1. Thermoregulation, that >
in: XI. Comparison with Clinical Situations and Predictiveness of the Tests it seems that the original work on the is cited:
As far as I have seen in the pdf :
http://www.farm.ucl.ac.be/Full-texts-FARM/Meunier-1998.pdf
>.....50µl of a 5% formalin solution was injected into....< and
>....The 5% formalin solution was prepared by dilution with normal saline of the commercial 10% formalin solution...
Use 37% commercial solution (buffered as possible) and make your dilution in PBS 10mM pH=7,4 to avoid pH changes due to formalin polymerization.
I used it for fixation in rodent, it worked very well.
Good luck.
VM
Dear Wolfgang H. Muss,
Thank you so much for the information, they are really helpful in guiding me to a successful formalin test. I am surprised that even the commecially used Formaldehyde in water (37% or 10%) contains a good proportion of stablizer, the majority of publication on formalin test are based on this mixture. But again, should not we use a comparable concentration of the stablizer , i.e. methanol, in saline rather than normal saline as the control?
But on the other hand, I also find some flaws when you prepare formaldehyde solution from PFA powder. I always find some suspended substance after PFA is dissolved in the NaOH buffered solution, so the actually concentration is unknown. Another problem might come from NaOH, which probably contains ions (in regarding to the source of NaOH) that has unknown effects on the animal and probably perplexes the situation.
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Dear Ana Claudia Reis Schneider, Dear Virginie Mauro,
Thank you for sharing your protocol. I hope I will make the formalin test works.
Best wishes,
Jiahao
I am going to use formalin for pain induction for first time. Reading the question and comments have been very helpful. Thank you all for contribution