Sectioning mouse eyes Help!

Allan Colthart ac50 at le.ac.uk
Mon Mar 3 09:44:52 EST 1997


Kevin Rattray wrote:
> 
> I have been having some difficulties with mouse eye sectioning.
> I do not perfuse the mouse or fix the eye before I freeze it down in OCT.
> I find that once I have sectioned down to the eye, the sections
> (12-14 microns thick) become difficult to handle. Perhaps, if there's
> static or even without static, it's difficult to get the whole eye
> mounted onto the slide. The eye parts, like the lens, don't stick
> together very well with the rest of the eye and sometimes will be
> rearranged or  will fall out.
> Should I be perfusing the mice, will that make the eye parts more
> cohesive when sectioning? Or should they be fixed and if fixed would that
> interfere with Immunofluorescence?
> One more question where could I get mouse eye Antibodies, something that
> I would know would act as a  good control for Immunofluorescence.
--------------
I assume that you are sectioning the entire eye longitudinally through
the lens (front to back).

Eye tissue tends to fall apart, but this can be minimised by careful
freezing. Use iso-pentane cooled in liquid nitrogen until syrupy, then
drop the whole fresh eye into it. The eye should be frozen within 20
seconds. The eye can be transferred to a vapour phase liquid nitrogen
storage system.

To section, mount the eye in the orientation required using OCT onto a
piece of cork on the chuck. Build up an OCT support around the eye, but
not over it, freezing in liquid nitrogen. You should be able to cut eye
sections at 6-7 microns and pick them up on adhesive coated slides. At
this thickness, the various parts of the eye should stay together and a
very slow controlled cutting action will help. At 12-14 microns, you
will get dragging artefacts ie. holes in the tissue section.

Dry the sections on the slides using a cool fan for at least 1 hour,
then store at -70C or use immediately. Fix the sections briefly (10-15
mins) in acetone (if you use silane adhesive slides, be careful which
type you use). Carry out the immunofluorescence etc.

The only antibody I can think of that reacts consistently with eye is an
pan epithelial marker eg. cytokeratin (contact your local antibody
shop). This reacts with the retinal pigment epithelium.

If you have any other problems please contact me by e-mail on 

ac50 at le.ac.uk

Good luck and happy sectioning



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