Protocol for Whole Animal/ Isolated Organ Vascular Perfusion Fixation( of rats/mice )

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.