New Person and Need Help with Histology

[Cardio]

I have done Histology for about 5 years now and I have run into a problem with a fixative commonly used in my lab. (1% paraformaldehyde, 1% cacodylic buffer, and NaCl) The tissue is prefixed in solution for either 24 hrs to 1 week. I have also post fixed as well.

The problem is that in a simple H and E stain the 10 micron section (mouse heart) develops shirnkage between cells.

I have narrowed down the problem between my fix and the dehydration step with ethanol. I can't explain it but as soon as the slide enters 75% ethanol the tissue starts to look like swiss cheese. (FYI. Unfixed tissue does not do this)

My question is whether anyone of you know if paraformaldehyde or cacodylic buffer is incompatible with ethanol or have anyone of you noticed this with a more common fix 4% paraformaldehyde?

[richard03]

Maybe due to the 75% ethanol you are using. Skip this and go directly to 95% ethanol.

I assume you are doing frozen sections. If so, the section needs to be air dried before staining.

[gula]

My first thougt is: isn't 1% too little for proper fixation?
If I remember right, in a study of Cecil Fox (1985), he found out, that there are no great differences between Formaldehyd-solutions from 2-20%, but under this line, the fixation-effect was reduced.
If I am right, your tissue, which contains an amount of collagen-fibers will undergo a dehydration-shock in the ethanol. The collagen-fibers shrink like a spring.
I would increase the Paraformaldeyhd (or Formaldehyd) amount to 4-8%.
gula

[excalibur]

I agree with gula. Richard's points are also true if fixation is good.

[Cardio]

My first thought is: isn't 1% too little for proper fixation?
If I remember right, in a study of Cecil Fox (1985), he found out, that there are no great differences between Formaldehyd-solutions from 2-20%, but under this line, the fixation-effect was reduced.
If I am right, your tissue, which contains an amount of collagen-fibers will undergo a dehydration-shock in the ethanol. The collagen-fibers shrink like a spring.
I would increase the Paraformaldeyhd (or Formaldehyd) amount to 4-8%.
gula

Thanks for all the responses guys. I have brought this to my boss's attention numerous times about the 1% paraformaldehyde being to weak. Unfortunately we are stuck with it for preserving our lacz signal and egfp signal in our transgenic hearts.

Increasing the concentration of paraformaldehyde will destroy our LacZ signal. I did however post fix our slides for 2 hours to address the shrinkage and it had little to no effect. On a 10 micron slide I figured 2 hours was long enough but perhaps not. Also it is very important to note that unfixed slides do not experience this dehydration shock. They in fact look incredibly good. If collagen in unfixed tissue looks preserved in the same H&E solutions then why does prefixed pfa tissue look terrible?

Last question for you guys. Have any of you noticed shrinkaged with 4% pfa prefixed tissue? I believe it is a common observation with frozen tissue but not in paraffin embedded tissue.

[gula]

This is only a assumption : It could be, that the muscle-cells and the collagen-fibers are in different states of fixation and therefore react in different sensitivity to the ethanol. Collagen fibers usually need a very good fixation to stay in place and don't shrink. - I think of skin-biospies, where one can see the gaps between the shrinked fibers. - And also due to the small amount of formaldehyde, it is likely, that the fibers first undergo a swelling in the aequous solution.
The muscle-cells should be fixed faster / better (?).

In the native frozen section, the parts of the tissue are in a equal situation.- no fixation at all.
Have you postfixed native sections or prefixed?

gula

[Cardio]

This is only a assumption : It could be, that the muscle-cells and the collagen-fibers are in different states of fixation and therefore react in different sensitivity to the ethanol. Collagen fibers usually need a very good fixation to stay in place and don't shrink. - I think of skin-biospies, where one can see the gaps between the shrinked fibers. - And also due to the small amount of formaldehyde, it is likely, that the fibers first undergo a swelling in the aequous solution.
The muscle-cells should be fixed faster / better (?).

In the native frozen section, the parts of the tissue are in a equal situation.- no fixation at all.
Have you postfixed native sections or prefixed? gula

So in non-transgeneic hearts processed and prefixed the same way as the transgenenics you get the same level of shrinkage. On a 10 micron section post fixed the sample looks pretty good.

Some lab members of mine have noticed a loss of collagen fibers in a fast-green siruis red stain using the fixative. The fix in my opinion appears to affect collagen and cell alike. Increasing the paraformaldehyde is a no can do because of the stability of LacZ. Postfix is also not an option with eGfp being water soluble. I just think I am screwed no matter what.

Not the first time and certianly not the last.

[ImmunoNYC]

Are your transgenic mice simultaneously transgenic for GFP and LacZ or is this two different mice. I am apt to argue your lacZ is already affected by the 1 percent PFA so the protocol isn't optimal regardless.

Can you remove the heart and cut in half? Half for snap freezing for lacZ (can be post fixed after cryosectioning in GA)  and the other half fix in 4 percent PFA?

[ImmunoNYC]

Another thought ...  you can do anti-beta gal IHC or IF and avoid having to do XGal enzymatic staining altogether, in which case you can fix properly in PFA.