I perfused some mice for IHC last week and found today that the pH of PFA was not adjusted to 7.4 (the pH is 11.3)
Is there anything I can do to save these brains and use them further for IHC or will it not work?
Dear Lukasz,
I think, you just have sections an try your immunohistochemistry. It is hard to believe that you have got a PFA solution with pH 11.3
Regards,
Fred
Hi Fred,
Thank you for your reply. The brains are still intact and in PFA at 4 degrees Celsius. The mistake I made was that I did not adjust the pH of PFA to 7.4 after clearing it with NaOH during preparation, therefore such basic pH.
My question is whether is it still worth trying to bring down the pH of these brains to 7.4, or would the proteins be already severely damaged?
Dear Lukasz, I would be interested in the procedure of your making PFA with an end-pH of 11.3. This is why I second Prof. Sinowatz' opinion " It is hard to believe that you have got a PFA solution with pH 11.3 ".... In my experience such only can happen if you used your freshly made PFA-solution (using NaOH for proper dissolution of the PFA-powder in slightly heated=approx. 65-70°C warm 'water' WITHOUT adding afterwards appropriate BUFFER solution (usually Na-CAcodylate or -as reported - better Na-Na-Phosphate buffer pH 7.2-7.4 (e.g. Millonig's PO4 buffer) with the right tonicity .... so: how did you actually make your 'final' perfusion solution ?? (Having done perfusion fixation of rat brains in my early "academic career", and mixed up - say at least 500 times - fresh and buffered PFA-solution for fixing human surgical specimens for diagnostic purposes I have the / a correct recipe in hands....)...
Dear Wolfgang,
Thank you for your extensive comment - very much appreciated!
As it was a very busy week, nobody checked the end-pH (it was never adjusted to 7.4 after clearing with NaOH!!).
The PFA we prepare is in dH2O at 65 Celsius, add PFA powder, clear with NaOH, allow to reach room temperature, add PBS component, adjust the pH to 7.4, and filter.
Lukasz.
Dear Sebastian,
Thank you for your comment. I have already processed with a new batch of animals at correct pH. I will include a few sections of the highly alkaline tissue in my next experiment as a control condition, to assess whether this affects our antibodies (we antigen retrieve with citric acid, maybe that would help the tissue, too?)
I will share a micrograph once it is done!
Lukasz
Lukasz: Good decision to try at least IHC-application to few sections of the "highly alkaline fixed tissue" for a comparison, I think.
Thank you also for the details you provided... But the essential part unfortunately you left out: "how much of NaOH [=drops from a pipet, ml...., and at which concentration, e. g. 0.1, or 1N or even 10N ] was added to the hydrous solution containing initially the unsolved PFA powder. In my hands-on experience it was only 2-3 drops (drop after drop with pause for mixing well! ) of 1N NaOH from a 1.5 ml one-way plastic pipet to add to 100 ml and the white turbulence cleared totally…
I refer to an old thread in RG, https://www.researchgate.net/post/I_should_prepare_0_1_M_PB_4_Paraformaldehyde_in_0_1_M_PB_and_0_1M_PBS_for_rat_brain_fixation_and_H_E_staining_How_can_I_prepare_these_buffers? , where the following statement was included (make of [theoretically] 40% hydrous PFA-solution, e.g. for further use in fixation, diluted with an appropriate PO4-buffer ]:
SAFETY NOTE:
wear at least gloves and safety googles, avoid to breath powder dust! Use a 250 ml beaker glass, put in first approx. 70-80 ml (water)=A.dest/A.bidest
40g PFA-powder, weigh out, place it - slowly - into - say 80 ml of A. bidest., stir a bit (powder will not dissolve completely).... ‘heat’ the solution carefully with [permanent] mixing (use magnetic stirrer) up to 60-70° (latter is the maximum!) and add drop by drop (!, from a 1.5-2.5 ml one-way-plastic pipette) slowly 1 N NaOH UNTIL the solution gets : Do not overtitrate. Dissolve left remnants of powder on the glass/beaker wall. Let cool to room temp. (eventually also filter the solution) and fill up with A. bidestillata to 100 ml (endconcentration = around 37-[40%]).
Best wishes and regards!
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