I am presently using 10% formalin for fixation of the micro-dissected tissues. They are 300µm in height and a diameter of approximately 420µm. We are presently trying various test at different incubation times to see which one is best in order to maintain a sufficiently good morphology quality. We have been asking ourselves if paraformaldehyde is better or not.
These tissues are very delicate and for that we want to minimize lose of cells and tissue decay.
Hi Kayla in the literature these two fixatives are often used interchangeably although the Formalin sometimes has ~5% v/v Methanol in to stabilise the formaldehyde
http://microscopy.duke.edu/sampleprep/formaldehyde.html
Methanol can have unexpected effects such as quenching GFP fluorescence (and at high concentrations it will permeabilise the cells)
The fixation time will probably have more impact than the
Both formalin and Para-Formaldehyde will penetrate tissue at ~ 1 mm per hour, so for such as small section, 15 minutes should be suitable to fix the section. Personally I would not fix your sections longer than 1 hour as this may have a detrimental effect on epitopes etc (I had the devil of a time performing ab staining on lung tissue that had been fixed overnight in Formalin.
Obviously it all rather depends on the downstream processing - if using histological dyes over fixation will not have such a detrimental effect.
I hope this helps and good luck with your work
Gary
Hi Kyala,
I think you can use paraformaldehyde in the form of Zamboni solution. It may give better result than that of 10% formalin. As your sample is very thin, your experience tells you how much time is needed.
Goodluck
Nazrul
Hi Kayla,
I basically agree with the previous answers. It is really a matter of what you want to do with the tissue after fixation. Mainly whether you want to do IHC (for either optic or EM) or standard stains. In "principle", formaldehyde would be a gentler fixation than paraformaldehyde. Generally for IHC I would recommend paraformaldehyde. And for certain IHC protocols and stonger fixation adding up to final concentration of 15 % (or lower) of a saturated picric acid solution (Zamboni) may work really well. For electron microscopy you can add glutaraldehyde up to a 0.05 % final concentration. For certain IHC protocols (e.g. using antibodies generated against a small molecule linked to a bigger one (albumin) via glutaraldehyde) you may hava to use glutaraldehyde up to a 1.25 % final concentration.
Having said that, formalin (formaldehyde) fixation may also work wonderfully for certain purposes. But at any rate I would use a pH 7 buffered and methanol stabilized formalin solution. It is commercially available and not too expensive.
I hope this is of certain help!
Best of luck!
Better PFA than Formalin, Better 4% than 10 % and finally I agree with Gary that you shold fix for short time. My samples are delicate tissue for about 7-9 mm Х 1-2mm
and I explore fthe ixation for 2-4 hours at +4-6 degree. The time of fixation is obviousely depends on the further procedure - for ICC the less time of the fixation - the beetter staining shlud you have, For just routine histological stsnings the time could be much longe and the comncenration can be 10 %. Good luck, Natalia
Thank you all, I am taking all your comments and recommendations into affect! fingers crossed!
For PFA preparation, what pH of PBS and final 4% PFA solution is best for tissue fixation?
Your are very welcome!
I would recommend a pH 7.2 -7.4
Good luck!
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