[Histonet] Rat perfusion protocol- what's wrong?

[Josephine Garcia]

Hi all,
We are trying to harvest muscle and nerve tissue from Sprague-Dawley rats
(upper leg muscle, sciatic nerve).
Is it necessary to perfuse the animal before harvesting muscle / nerve, or
will simply soaking in 4% PFA do?
Our perfusions often, but not always, fail. By "fail", I mean the rat does
not twitch after Paraformaldehyde is circulated, and the tissue does not get
stiff. Here is our protocol.

·         Inject with 0.5 mL Beuthanasia (pink liquid, draw up with 3mL
syringe) into liver (right abdomen)

·         Open chest cavity on perfusion table by pulling skin on chest up
and cutting away circular region. Pierce diaphragm, cut up through ribs
(towards armpit) with blunt side of scissors down, exposing heart and lungs

·         Inject butterfly needle (connected to pump) into the lobe that
faces the back – go up into the heart from the bottom (see diagram)

·         Turn on perfusion pump (clockwise button) at ~20mL/min

·         Cut (using smallest scissors available) black sac near the heart –
this is the vena cava. If done correctly, the cavity should fill with blood

·         Pump > 60mL of saline from the syringe into the system, draining
the blood. Once the blood being drained turns into a clearer red, switch to
Paraformaldehyde

·         Pump > 150mL of cold 4% Paraformaldehyde. The rat should twitch
and stiffen within minutes if perfusion is successful (twitching occurs ~90%
of the time)


Also, if perfusion is successful, we soak the tissue in 0.4%
paraformaldehyde instead of 4%. Is this correct?


Thanks for the help.
**