Jul 23, 2020
Perfusion
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Molly Brennan, Olivier George 2020. Perfusion. protocols.io https://protocols.io/view/perfusion-bivzke76
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 22, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 39577
WHOLE ANIMAL FIXATION VIA TRANSCARDIAL PERFUSION
List of Stuff Needed for Perfusion
-4% Formaldehyde
-0.9% Hep-saline
-Bottles & pump
-Surgical tool (scissors, clamps, bone rongeurs, spatula)
-Vials
-timer
Preparation
-Switch waste flow to deposit into chemical waste bucket
-Prepare perfusion pumps with 0.9% saline and 4% paraformaldehyde, check flow
-Test system; check for flow and air bubbles
Anesthesia
-Inject Clorohydrate 35 % into the left lower ventral abdomen
-Wait approximately 5 min for anesthetic to take effect
-Pinch the tip of the tail forcefully to test for reaction
-Add additional amount of anesthesia if necessary
Perfusion Rates:
Koob Lab—Pump runs in RPMS
-Prefixative rinse -saline- 3 minutes @ max 90 RPM(i.e. ~24mls/min)
-Formaldehyde- 5 minutes @ max 90 RPM(i.e. ~24mls/min)
-Continue Form- 15 minutes @ 65 RPM(i.e. ~16mls/min)
Surgery and Fixation (do under hood or safe place)
-Place animal on the downdraft, make a “V” shape above the genital
-Lift skin upwards firmly using forceps, pinch a small hole into the abdomen, then cut along the “V” line
-Continue to cut upwards to expose the diaphragm, and then cut through the diaphragm
-Cut through the rib cage to expose the thoracic cavity
-Clamp open excess tissue for better access of the heart
-While holding the middle of the heart steady with forceps, make a cut slightly right to the apex of the heart (left ventricle), using one tip of a sharp scissors, then insert both tips of small scissors to make a bigger hole
-Insert catheter needle through the cut, up to the aorta. Clamp the entrance
-Make a cut in the dark red-colored right atrium,
-Circulate 0.9% saline, make sure fluid is flowing freely, until the limbs of the rat are discolored (3 min)
-Turn off saline pump, and circulate 4% paraformaldehyde (approximately 250 ml) until neck is stiffened (3+15 min)
Brain Removal
-Use a pair of scissors to cut connective tissue under the skin, reaching in between the eyes
-Cut skin open, exposing muscles on top of skull to the eyes
-Use scalpel, cut muscle connecting to the skull bone on both sides
-Carefully place bone cutter against the inside of the skull, and make cuts following the scalpel incision
-Lift the occipital, parietal, and frontal bones using forceps, fully exposing the brain
-Cut membrane on top and scoop brain out of the skull; cut optical nerves and connective tissue
-Store brain in 4% fixative for 1 day.
Sucrose Treatment
-Take the brain out of the fixative, dab dry on utility towel
-Place brain in 20% sucrose solution (in distilled water) until it sinks to the bottom of the vial
-Place brain in 30% sucrose solution (in distilled water) for another day until it sinks to the bottom of the vial
-Freeze brains before cutting in dry ice.