[Zbrafish] Zebrafish Embryo Fixation

Bill Trevarrow via zbrafish%40net.bio.net (by Trevarrow from EugeneResearchAquatics.com)
Tue Nov 23 15:09:45 EST 2010

Hello Harry, 

I expect that your embryos look black because your are illuminating them with transmitted light (light from below, going through the specimen before it gets to your microscope). 
Normally fixed tissue will be opaque white. It will look white in reflected light (using a light source above the specimen so the light is reflected to the microscope). Because the embryo is opaque, little or no light is able to get through the specimen to view, paradoxically resulting in a black looking embryo, even though it is white (in reflected light). 

The embryos can be cleared by running them through a graded series of inccreasingly more concentrated glycerol solutions, up to 50 or 100% glycerol. 

The small particles you saw when dechorionating, were most likely fixed yolk granules, which can come loose if the yolk cell is disrupted. 

Many people will dechorionate their embryos before fixing to provide the fixative better access to the embryo. 

Depending upon the age of the embyro and the particular labeled structures, some people dissect away the yolk or other parts and may flatten out the embryo to more easily visualize the labeling patterns. 

You might want to consider using a larger volume of fixative if your specimens are not sufficiently well fixed. 

On Nov 23, 2010, at 12:36 AM, Harry wrote:

> Dear friends,
> I am working in Zebrafish and I am a beginner. My interest is in whole
> mount ISH. I am suppose to make an atlas of developmental stages of
> the embryos at first as a part of standardization of fixation and etc.
> I am trying to do a small scale experiment. I put 3-4 embryos at
> particular time intervals in 150 microlitres of freshly prepared 4%
> Paraformaldehyde. After keeping it in 4 degrees  for 8-10 hours, I try
> to take the photograph of the fixed embryo. I put the embryo in a
> petridish with 1X PBST and dechorionate that under a stereomicroscope.
> The embryo turns black (especially the yolk) after dechorionation. If
> further disturbed, the yolk disintegrate into small cell like
> structures. I would like to know the reason for the happening and
> humbly ask for suggestions to correct or improve my procedure of
> fixation.
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Bill Trevarrow, Ph.D.
Eugene Research Aquatics, LLC
Trevarrow from EugeneResearchAquatics.com
Tel: (541) 844-9054
Fax: (541) 683-0996

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