Stentor

Images made through a microscope. All subject types.

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blepharopsis
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Stentor

Post by blepharopsis »

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.

Image

Image

Jacek
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Post by Jacek »

:shock: :shock: :shock: :shock: :shock: WONDER !

Olympusman
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Stentor

Post by Olympusman »

Amazing!

Mike
Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA

zzffnn
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Post by zzffnn »

Wow! Great professional work and amazing images. This is Nikon Small World material. Igor, have you sent them for an image competition?

blepharopsis
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Post by blepharopsis »

Thanks!!
Zzffnn - as a matter of fact, yes - but not exactly those two.

75RR
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Post by 75RR »

striking images!
Zeiss Standard WL & Wild M8
Olympus E-p2 (Micro Four Thirds Camera)

WalterD
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Post by WalterD »

Stunning images, Igor. Innovating, creative and visually overwhelming.
Hope too see much more :)

Harald
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Post by Harald »

Hi blepharopsis,
This is great.
Exceptional images. Love the last one.

Thanks for sharing :D
Kind Regards
Harald

Lier Fotoklubb / NSFF
AFIAP / CPS
BGF / GMV
http://www.500px.com/blender11

RogelioMoreno
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Post by RogelioMoreno »

Igor,

Wow, amazing images!
The second one was with 40x too?

Rogelio

Smokedaddy
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Post by Smokedaddy »

What else can I say but excellent.

pwnell
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Post by pwnell »

Never seen this level of clarity and detail of this fragile species before.

Robert Berdan
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Truly amazing photos

Post by Robert Berdan »

Very impressive images of Stentors - I will give the technique a try.

Thanks for sharing.
RB

Marek Mis
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Post by Marek Mis »

Igor, your confocal images are from different plannet ! Fabulous !
Thank you for sharing.

Regards,

Marek

carlos.uruguay
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Post by carlos.uruguay »

Marvelous!!!!!!!!!!!!!!!!!!!!!

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