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Stentor

 
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blepharopsis



Joined: 28 Nov 2014
Posts: 143
Location: Virginia, USA

PostPosted: Fri May 17, 2019 9:17 am    Post subject: Stentor Reply with quote

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.



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Jacek



Joined: 02 Oct 2011
Posts: 5342
Location: Poland

PostPosted: Fri May 17, 2019 9:23 am    Post subject: Reply with quote

Shocked Shocked Shocked Shocked Shocked WONDER !
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Olympusman



Joined: 15 Jan 2012
Posts: 4229

PostPosted: Fri May 17, 2019 9:34 am    Post subject: Stentor Reply with quote

Amazing!

Mike
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Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA
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zzffnn



Joined: 22 May 2014
Posts: 1818
Location: Texas USA

PostPosted: Fri May 17, 2019 10:04 am    Post subject: Reply with quote

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



Joined: 28 Nov 2014
Posts: 143
Location: Virginia, USA

PostPosted: Fri May 17, 2019 10:09 am    Post subject: Reply with quote

Thanks!!
Zzffnn - as a matter of fact, yes - but not exactly those two.
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75RR



Joined: 22 May 2014
Posts: 674
Location: Estepona

PostPosted: Sat May 18, 2019 6:55 am    Post subject: Reply with quote

striking images!
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WalterD



Joined: 06 Jul 2015
Posts: 523
Location: Rotterdam, the Netherlands

PostPosted: Sun May 19, 2019 2:40 am    Post subject: Reply with quote

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



Joined: 13 May 2011
Posts: 570
Location: Steinberg, Norway

PostPosted: Sun May 19, 2019 4:33 am    Post subject: Reply with quote

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

Thanks for sharing Very Happy
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Harald

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RogelioMoreno



Joined: 20 Nov 2009
Posts: 2962
Location: Panama

PostPosted: Thu May 23, 2019 10:26 am    Post subject: Reply with quote

Igor,

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

Rogelio
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Smokedaddy



Joined: 07 Oct 2006
Posts: 1437
Location: Phoenix, Arizona

PostPosted: Thu May 23, 2019 3:09 pm    Post subject: Reply with quote

What else can I say but excellent.
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pwnell



Joined: 18 Dec 2009
Posts: 2000
Location: Tsawwassen, Canada

PostPosted: Mon May 27, 2019 12:54 pm    Post subject: Reply with quote

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



Joined: 18 Oct 2017
Posts: 150
Location: Calgary

PostPosted: Tue Jun 04, 2019 9:26 am    Post subject: Truly amazing photos Reply with quote

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

Thanks for sharing.
RB
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Marek Mis



Joined: 10 Jul 2010
Posts: 2560
Location: Suwalki, Poland

PostPosted: Tue Jun 11, 2019 1:54 am    Post subject: Reply with quote

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

Regards,

Marek
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carlos.uruguay



Joined: 23 Feb 2012
Posts: 5249
Location: Uruguay - Montevideo - America del Sur

PostPosted: Thu Jun 13, 2019 2:47 pm    Post subject: Reply with quote

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