Stentor

blepharopsis · Post by **blepharopsis** » Fri May 17, 2019 9:17 am

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.

Jacek · Post by **Jacek** » Fri May 17, 2019 9:23 am

WONDER !

Olympusman · Post by **Olympusman** » Fri May 17, 2019 9:34 am

Amazing!

Mike

zzffnn · Post by **zzffnn** » Fri May 17, 2019 10:04 am

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

blepharopsis · Post by **blepharopsis** » Fri May 17, 2019 10:09 am

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

75RR · Post by **75RR** » Sat May 18, 2019 6:55 am

striking images!

WalterD · Post by **WalterD** » Sun May 19, 2019 2:40 am

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

Harald · Post by **Harald** » Sun May 19, 2019 4:33 am

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

Thanks for sharing

RogelioMoreno · Post by **RogelioMoreno** » Thu May 23, 2019 10:26 am

Igor,

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

Rogelio

Smokedaddy · Post by **Smokedaddy** » Thu May 23, 2019 3:09 pm

What else can I say but excellent.

pwnell · Post by **pwnell** » Mon May 27, 2019 12:54 pm

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

Robert Berdan · Post by **Robert Berdan** » Tue Jun 04, 2019 9:26 am

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

Thanks for sharing.
RB

Marek Mis · Post by **Marek Mis** » Tue Jun 11, 2019 1:54 am

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

Regards,

Marek

carlos.uruguay · Post by **carlos.uruguay** » Thu Jun 13, 2019 2:47 pm

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

Stentor

Stentor

Stentor

Truly amazing photos