Advantages:
• Fixation begins
immediately after arrest of systemic circulation. This
minimizes the
alteration of cell
structure resulting from post-mortem effects.
• Under in
situ conditions, vascular
perfusion results in
a uniform and
rapid
dissemination of
fixative into all parts of the tissue
via the vascular bed, resulting
in an
increased depth and
rate of actual fixation.
• The
manipulation of tissues after the
arrest of the systemic
circulation but prior
to
fixation is minimized
resulting in many fewer artifacts.
• Many organs/
tissues may be effectively fixed at one time, thus maximizing the use
of
each animal.
• In the
case of immunocytochemical procedures
employing relatively mild fixation
conditions, fewer
autolytic artifacts result;
redistribution or translocation
of cellular
components is
minimized; and greater immunocytochemical activity is retained.
Pre-perfusion:
1. The rat/mouse
is retrieved from the
animal quarters and
brought upstairs to the
surgery room in a
cage. The person removing the
animal should sign
and date the
animal inventory card
( yellow card ) in the out column.
2. The rat/mouse
is weighed and
injected with a
mixture consisting of
ketaset(75
mg/ml) + xylazine(5
mg/ml). The recommended injectable dose
of this anesthetic is 1
ml/gm of body weight,
IM. Allow 10 to 15 minutes for
anesthesia to occur, indicated by
the loss
of sensory/ reflex
response, i.e. non-
response to tail
pinching, or paw
pinching. (Note:
Metofane, as an inhaled
anesthetic, may be
used instead of the
ketaset/ xylazine
mixture. In this case, assistance of another person is advised.)
3. Once anesthetized,
and during the surgical procedures for whole
animal or isolated
organ perfusion,
care should be taken to
prevent heat loss
in the anesthetized
animal. They are quite prone to hypothermia.( use of a lamp is
recommended )
4. People should
consult Michael for the perfusion method for their particular needs. In
general, an isolated
organ perfusion will yield the best results, but concern
should be
taken to ensure
maximum, most efficient use of the rat/mouse.
For example, in the
case of an isolated
perfusion of the heart/lungs, the
rest of the animal
might still be
suitable for
biochemistry.5. In most instances, the rat will expire during the perfusion. However, in cases where
the animal survives
or lingers, cardiac puncture is the appropriate means of sacrifice.
6. Upon
completion of the
procedure the carcass
is wrapped in
a surgical pad/
or
benchcoat, placed in
a plastic bag, and returned to the
animal quarters in
the cage.
The carcass is placed
in the freezer; and check that the
animal inventory card ( yellow
card ) is signed off.
Recommended routes
for vascular perfusion:
• Whole animal-
descending aorta or vena cava.
• Central nervous
system/ pituitary- aorta, via the left ventricle.
• Kidney- descending
aorta, proximal to its distal bifurcation.
• Liver- portal vein
Perfusion Protocol
Perfusion pressure,
in most instances,
should be maintained
between 60 and
100
mm Hg. Use a
sphygmomanometer or gravity feed apparatus... we have both!!!
1. Generally, it is
optimal to aerate/oxygenate the flush and fixation prior to beginning.
This can be
maintained during the perfusion.
2. Flush the
animal/organ first with 1XPBS containing
1% sodium nitrite, pH 7.4 for 30
seconds.
3. Follow up flush
with perfusion of fixative for 5 minutes. Fixative contains:
3% formaldehyde
(freshly prepared from paraformaldehyde);
1.5% glutaraldehyde
2.5% sucrose
contained in 100mM
cacodylate, pH 7.4
200mls of fixative
should be plenty for a mouse; 500 mls for a rat.
4. After 5 minutes
of continuous perfusion,
organs can be
harvested; appropriately
dissected in
fixative/buffer (100mM cacodylate,
2.5% sucrose, pH
7.4); and tissue
pieces allowed to fix
an additional 1 hour.
5. Wash in 0.1M
Cac/2.5% sucrose pH 7.4 3X 15' EA.6. Post-fix with Palade's OsO4 for one hour
on ice, light tight, under hood.
5 ml Palade's 1% OsO4
= 1 ml Acetate-veronal stock
+ 1.25 ml 4% OsO4
+ 1 ml 0.1N HCl
+ 1.75 ml ddH2O
Acetate-veronal stock
= 1.15 g NaAcetate Anhydrous
(J.T. Baker 1-3470)
+ 2.943 g
NaBarbituate (Veronal)
labelled
Barbital--(Sigma B-0500)
to 100 ml with ddH2O
7. Rinse 1X with
Kellenberger, then incubate for 1 to 2 hrs. at RT. (or preferably
overnight)
10 ml Kellenberger =
2 ml Acetate Veronal Stock
+ 2.8 ml 0.1N HCl
+ 5.1 ml ddH2O
+ 0.05 g Uranyl
Acetate
Check pH with paper
before adding UA.
(Should be ~6)
8. One quick rinse in
ddH2O, then one quick rinse in 50% ethanol.
9. Dehydrate with
graded series of cold (4
0
C) ethanol (70, 95,
100); then three 10’
washes in fresh 100%
ETOH at rm. temp; and then finally two 5’ exchanges with
propylene oxide (PO).
14. Place in 50%
PO/50% Epon (can be old) overnight, uncovered under vacuum (or
hood).
10. Replace with
fresh, 100% epon, and leave under vacuum for 2-6 hours.
11. Embed in fresh,
100% Epon. Put typed or pencil-written label in dummy capsules
with wooden stick, at
least two capsules per sample. Pour tissue out of tube into
mincing dish. Place
tissue in flat mold with small amount of Epon (to avoid curling)
with a wooden stick
and place in 60o oven overnight.
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