Methanol for immunohistochemistry

Model Organism: Worms Methods & Reagents
1

What methanol (i.e. what Sigma catalog number) are people using for immunohistochemistry? I am staining embryos using a freeze-crack protocol followed by methanol and acetone fixation, but my microtubules (stained as a control) do not appear to be well-preserved.

Thanks,

Nancy

2

Nancy,

Any reagent grade 100% methanol should work for methanol-acetone fixation.

However, the anti-tubulin antibodies that I have used require the tubules to be fixed with formaldehyde.
They do not work with methanol-acetone fixation.
Are you sure that the antibody is supposed to work on methanol-acetone fixed tubes?
If not, you could try 1 hour of 2% formaldehyde (the fix I use).

Good luck,

Janet

3

Thanks for the reply. I’ve successfully used this anti-tubulin antibody with a methanol-acetone fix in another lab (with different methanol). The primary goal isn’t to stain for tubulin, but for another protein that doesn’t work with a formaldehyde fix. Since the microtubules don’t look good, it makes me wonder what else isn’t right with the fixed cells.

  • Nancy
4

Another potential variable might be the temperature of fixation?
Microtubules are unstable at low temp’s, and perhaps your recent problems correlate with a shift to a low temperature fixation?
Compare a room temp fix, rather than fixation at 4 degrees, and see if that makes a difference?

Dave

5

Dave - Thanks for the suggestion. I may give it a try.

  • Nancy
6

Nancy,

Another thought, since I have used ice cold fix on tubulin and have not had a problem.
(I think the fix fixes the tubes before they have a chance to fall apart due to the cold.)
One possibility is that your anti-tub antibody recognizes an epitope that is poorly conserved in worms.
Another is that you are not getting the worms fully cracked.
Do you have DAPI in your mounting media and does that look great?

Good luck, Janet

7

Not sure which antibody you’re using. I am using the sigma DM1a anti-alpha tubulin antibody, and I crack and then fix in ice-cold MeOH for 8-10m (no acetone or PFA) with much success. But Ahna Skop once suggested using RT MeOH to avoid morphology distortions caused by the embryo shrinking.

Steve