Kathy,
I've permeabilized tissue (molluscan larvae and adult molluscan brains,
and locust CNS) in two ways. One is to run the tissue through and alcohol
series up to xylene and back down again (PBS, dH2O, dH2O, 30% EtOH, 50%,
70%, 80%, 90%, 95%, 100%, 100%, 100%, xylene, xylene, 100%, 100%, 100%,
95%, 90%, 80%, 70%, 50%, 30%, dH2O, dH2O, PBS). The amount of time per
step depends on the tissue and its size. For insect brains I'd start with
15 min/step. This procedure removes lipids and also, obviously, disrupts
the membranes of cells to a certain extent, but not enough to interfere
with seeing axonal processes and neuron cell bodies.
A second method I've used with some success is to prepare a solution of
PBS that contains 5% Triton-X100 (yes, I really said 5%) and soak the
tissues in this solution overnight in the refrigerator and then rinse the
tissue in PBS. I've used this on molluscan larvae with excellent results.
These larvae are about 200 um, so for larger pieces of tissue it might
take a longer soak. You'll just have to try it and see.
What you might want to do to determine the lengths of time tissues should
be treated is to label some treated tissues with an antibody that binds to
known structures in the ganglia. Anti-5HT or anti-SCP would work well
with insect ganglia. This would give you a good idea of how long the
tissues should be soaked in each alcohol step or in PBS with 5% triton.
Good luck,
Steve
_________________________________________________________________________
The mind is like a parachute; it works much better when it's open.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Stephen C. Kempf Tel: 334-844-3924
Associate Professor Fax: 334-844-4065
Depart. of Zoology and Wildlife Science
101 Cary Hall
Auburn University, AL 36849
USA Email: [log in to unmask]
Director - AU Hybridoma Facility - www.auburn.edu/research/hybridoma/
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On Fri, 13 Nov 1998, Katherine Terry wrote:
> I'm trying to fix whole insect brains (Rhodnius prolixus) using 4%
> paraformaldehyde and I need information about which detergents work best
> to permeabilize the tissue to improve antibody penetration. Different
> sources offer conflicting info on what should work best. Help! Does
> anyone have a preference? I've come across Triton X-100, Tween 20, and
> Saponin. Any advice will be greatly appreciated!
>
> Thanks,
>
> Kathy T
>
> Katherine Terry
> [log in to unmask]
> York University - Biology Department
> Farquharson 010
> 4700 Keele St
> Toronto, Ontario
> M3J 1P3
> CANADA
>
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