Brain Ischemic Injury in Rodents: The Protective Effect of EPO - Tissue-Protective Cytokines: Methods and Protocols - page 88

Comment: If fluid is coming from animal's nostrils or mouth,
reposition the cannula.
16. Turn on the pump to allow perfusion with the heparinized saline
and at the same time perform an incision in the right atrium.
17. When blood has been cleared from the body, change to fixative.
Take care not to introduce air bubbles while transferring from
one solution to the other. To avoid air bubble coming through
turn off the peristaltic pump while moving the tube to the
other solution and then turn it on again. The perfusion with
PFA solution will induce muscle spasms (“formalin or death
dance”) of the whole body attesting a good perfusion.
18. Perfusion is almost complete when those spontaneous move-
ments and lightened color of the liver are observed.
19. At the end or before perfusing another animal clean the tube
and fill it again with heparinized saline. Avoid injection of PFA
residue first.
20. Dispose of paraformaldehyde waste into specific containers,
and wash glassware and all tools extensively.
21. Perform a decapitation and put the whole head (open the back
of the skull to allow a good post-fixation of the brain) in 4% PFA
in 0.1 M PB for post-fixation during 24-72 h or extract just the
brain, remove the dura mater, and post-fix it in the same way. After
that you can put the brain in sucrose solution and then slice it.

Section the brains coronally in 20-40 mm slices using a cryostat
(Leica 3050 S), a freezing microtome, a microtome or a vibratome
depending of the sample method preparation.
Mount sections on gelatin coated or positive charged plus
slides (SuperFrost Ultra Plus ® , SuperFrost Plus ® ; Menzel-Glaser).
Air-dry sections during 2 or 3 days or bake slides on a slide warmer
overnight.

Cryosectioning
and Staining

Stain as follows:

Staining Protocol for Fresh-Frozen Sections

1. 96% Ethanol: 1¢
2. 70% Ethanol: 1¢
3. 50% Ethanol: 1¢
4. Distilled H 2 O: 1¢
5. Thionin Stain: 5 ¢ (see Note 8)
6. Distilled H 2 O: 30 ″

7. Distilled H 2 O: 30 ″

8. 50% Ethanol: 30 ″

9. 70% Ethanol: 30 ″

10. 96% Ethanol: 30 ″