I would appreciate your protocole for glucose Oxidase method.
Concerning the shrinkage there was a reply which sent me to a link with the
"perfusion one" apparatus, with the following info about shrinkage:
The shrinkage occurs when a prewash with physiological saline is followed
by fixative. The first action of fixative on the cell membrane is to shut
off the sodium pump proteins. Sodium rushes into the cell, followed by
water to maintain tonicity, and cells swell and expand. Membrane proteins
are fixed and crosslinked to neighboring cells in the swollen position.
Later, the cells stabilize and contract, and pull other cells with them.
The result is gross shrinkage, local distortion and torn membranes with
lost cellular contents from some cells.
Tissue Shrinkage can be completely prevented if the extracellular fluid
with ions is replaced by a nonionic isotonic solution that cannot enter the
cells. This can be accomplished by prewash with 5% (isotonic) sucrose in
distilled water. Start the prewash at low pressure, and pump up to 300 mmHg
to break the blood brain barrier and wash the extracellular fluid (and red
blood cells). Switch shortly thereafter to fixative. The perfusion takes
the same time as the traditional procedure, and accomplishes the favorable
results bulleted above.
What do you think about this?
After perfusion of the mother I make a sagital incision of each embryo (so
the fixative an inclusion infiltrates) then postfix in PFA overnight and 4h
in PBS. My inclusion protocole is the same that I use for whole brain from
adult rat and mouse:
Alcohol 70 - 4h
2x Alcohol 95 - 2h
3x ETOH - 2 h
2x Clearify (xylene substitute) - 2h
3x paraffin - 2h.
Thanks again for your advice,
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