germinal vesical staining in granulosa enclosed oocytes

[Tasha Czarny]

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Hello

I am trying to stain enzymatically dissociated ovarian follicles to
determine the oocytes viability using SYBR14 and propidium iodide. I am
working in a marsupial which has only one layer of tightly bound
cumuls/granulosa cells and I know from previous experiments that the stain
can penetrate the egg.

The surrounding cells stain brightly but I cannot find the GV when I assess
with confocal- either by making a z-stack or manually scrolling.

Does anyone have any hints on how to see the GV, it should be large and
obvious, but my oocytes appear empty with only staining in the surrounding
granulosa cells.

Thanks in advance for any help

Tasha Czarny
PhD student
Marsupial Research Lab
The University of Newcastle, Australia

[Dariusz Maluchnik]

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Try another nuclear stain, eg. Hoechst/DAPI (needs UV or 405 nm diode
laser) or DRAQ5.

Darek Maluchnik

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[Khaled Machaca]

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Tasha,

Don't know about marsupials, but in xenopus when we stain the oocyte  
one cannot see the GV because the DNA is too diffuse so you don't get  
a good signal.  One the chromosomes condense after GVBD they are easy  
to detect.


On Sep 1, 2008, at 10:11 AM, Tasha Czarny wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello
>
> I am trying to stain enzymatically dissociated ovarian follicles to
> determine the oocytes viability using SYBR14 and propidium iodide.  
> I am
> working in a marsupial which has only one layer of tightly bound
> cumuls/granulosa cells and I know from previous experiments that  
> the stain
> can penetrate the egg.
>
> The surrounding cells stain brightly but I cannot find the GV when  
> I assess
> with confocal- either by making a z-stack or manually scrolling.
>
> Does anyone have any hints on how to see the GV, it should be large  
> and
> obvious, but my oocytes appear empty with only staining in the  
> surrounding
> granulosa cells.
>
> Thanks in advance for any help
>
> Tasha Czarny
> PhD student
> Marsupial Research Lab
> The University of Newcastle, Australia

-Khaled

Khaled Machaca, PhD
Associate Professor of Physiology & Biophysics
Associate Dean for Basic Science Research
Weill Cornell Medical College Qatar
Qatar Foundation - Education City
Tel: [phone]   Fax:  [phone]
[hidden email]

[Julian Smith III]

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I get the same result in flatworms with DAPI--until GVBD, the DNA is too
diffuse to get a good signal.
Julian

Khaled Machaca wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Tasha,
>
> Don't know about marsupials, but in xenopus when we stain the oocyte
> one cannot see the GV because the DNA is too diffuse so you don't get
> a good signal.  One the chromosomes condense after GVBD they are easy
> to detect.
>
>
> On Sep 1, 2008, at 10:11 AM, Tasha Czarny wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hello
>>
>> I am trying to stain enzymatically dissociated ovarian follicles to
>> determine the oocytes viability using SYBR14 and propidium iodide. I am
>> working in a marsupial which has only one layer of tightly bound
>> cumuls/granulosa cells and I know from previous experiments that the
>> stain
>> can penetrate the egg.
>>
>> The surrounding cells stain brightly but I cannot find the GV when I
>> assess
>> with confocal- either by making a z-stack or manually scrolling.
>>
>> Does anyone have any hints on how to see the GV, it should be large and
>> obvious, but my oocytes appear empty with only staining in the
>> surrounding
>> granulosa cells.
>>
>> Thanks in advance for any help
>>
>> Tasha Czarny
>> PhD student
>> Marsupial Research Lab
>> The University of Newcastle, Australia
>
> -Khaled
>
> Khaled Machaca, PhD
> Associate Professor of Physiology & Biophysics
> Associate Dean for Basic Science Research
> Weill Cornell Medical College Qatar
> Qatar Foundation - Education City
> Tel: [phone]   Fax:  [phone]
> [hidden email]
>

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

[Tasha Czarny]

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Thanks everyone,

I need to keep the same stains and can't increase the membrane permeability
as I'm looking an cell viability associated with membrane permeability.

I am going to try confocal with brightfield then transfer to the lasers once
in position, and maybe try to increase their strength

I'll post an update to let you know how it goes

Thanks for the feedback
Tasha