Stentor
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Stentor
Finally, I found a protocol that works pretty well for preserving swimming form of Stentor for immunohistochemistry - maybe with some tweaking I'll get a fully extended one. From Huang & Pitelka (J. of Cell Biol. 1973, Vol. 57): "Cells in the extended state were obtained (...) by treatment before fixation with a solution containing 10 mM EGTA, 50-80 mM Tris, 3 mM MgSO4, 7.5 mM NH4Cl, 10 mM phosphate buffer (pH 7.1)".
Staining - anti-acetylated tubulin mAbs (cilia - red);
DAPI (DNA - blue);
I also used actin marker Alexa 488-phalloidin, but's it's known to fail to recognize protozoan F-actin - the green channel is mostly a combination of bleed-through signals for both blue and red channels and some autofluorescence.
The first image (a compilation of 7 individual scans) shows stentors fixed with formaldehyde only - cells collapse a bit due to osmotic stress, some rupture, but the signal from surface cilia is strong. The second image - I used just a dash of glutaraldehyde - the preservation of the cell shape is better, but I feel that glutaraldehyde messes up with the exposed epitopes in surface cilia, judging from weaker signal. The bases of cilia still stain fine.
Zeiss LSM 880, 40x NA 1.3.
Staining - anti-acetylated tubulin mAbs (cilia - red);
DAPI (DNA - blue);
I also used actin marker Alexa 488-phalloidin, but's it's known to fail to recognize protozoan F-actin - the green channel is mostly a combination of bleed-through signals for both blue and red channels and some autofluorescence.
The first image (a compilation of 7 individual scans) shows stentors fixed with formaldehyde only - cells collapse a bit due to osmotic stress, some rupture, but the signal from surface cilia is strong. The second image - I used just a dash of glutaraldehyde - the preservation of the cell shape is better, but I feel that glutaraldehyde messes up with the exposed epitopes in surface cilia, judging from weaker signal. The bases of cilia still stain fine.
Zeiss LSM 880, 40x NA 1.3.
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- Robert Berdan
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Truly amazing photos
Very impressive images of Stentors - I will give the technique a try.
Thanks for sharing.
RB
Thanks for sharing.
RB
- carlos.uruguay
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