TITLE: Protocol for mouse perfusion with dye, DAPI staining, and slide preparation
AUTHORS: Boaz Mohar, Monique Copeland

[STEPS]
?. Add 50-100 ul of 1mM dye stock to  of 4% paraformaldehyde in .1M PB
[per mouse]
?. [Set-up hood for perfusion]
Set up fume hood with:
?. [Prepare dye and fix]
Add   of DMSO to lyophilized dye (100nmol) and Pipette up and down ~20 times to get 1mM concentration stock.
100 µl
?. Mix further using a vortex machine to ensure the dye is evenly distributed in the DMSO
?. [Perfusion and dissection]
Perfusion pump preparationAttach needle:to pump tubing and flush with ~  of 1x PBS  to clear tubing of previous solution
20 ml
?. [Perfusion and dissection]
Retreive animal per vivarium protocol
?. Prepare 4% Paraformaldehye in 0.1M PB see here
?. [Perfusion and dissection]
Anesthetize mouse in 2000ml glass beake
?. [Perfusion and dissection]
Place the animal inside the container on a raised platform and cover.(Platform can be square slides box lid or styrafoam from 50ml tube packaging)(DO NOT allow the liquid isofluorane to come into contact with the animals’ skin)
?. [Perfusion and dissection]
As soon as the animal stops breathing, remove from contain, check for pain response by gently pinching the foot with forceps and make sure no reflexive response and place on dissection tray.        Timing is extremely important.  If the animals is over-exposed to the isofluorane than the heart will stop beating and cell death can occur.
?. [Perfusion and dissection]
Begin perfusion
?. [Perfusion and dissection]
Lay mouse on its back and place a pin in each front paw, others as needed.
?. [Perfusion and dissection]
Use scissors to cut parallel to the sternum, opening the chest cavity and exposeing the heart.
?. [Perfusion and dissection]
Insert winged needle into the right ventricle.
?. [Perfusion and dissection]
Cut left atrium.
?. [Perfusion and dissection]
Start perfusion pump and flush  with   at a rate of 7.5ml/min.(Animal will "twitch" when the fixative circulates through the bloodstream.  If no reaction occurs check postion of the needle in the heart.)
[4% PFA]
[of Dye]
?. [Perfusion and dissection]
"Stop" perfuison pump when all fixative is flushed through the animal.
?. [Perfusion and dissection]
Unpin mouse from dissection tray and decapitate behind ears.(Place carcass in small biohazard bag for disposal.)
?. [Perfusion and dissection]
Remove the fur from the head and cut small slits into the skull by placing scissor tip on eithier side of the opening where the spinal cord connect to the brain.
?. [Perfusion and dissection]
Use forceps to peel off skull cap and scope out brain.
?. [Perfusion and dissection]
Post-fix brain in  at
[4% PFA]
4 °C
?. [Perfusion and dissection]
Rinse 3 times  in PBSat
0 Room temperature
?. [Perfusion and dissection]
pour  isoflorane down the side of the container.
5 ml
?. [Prep brain for sectioning]
Prepare
[4% agarose]
?. [Prep brain for sectioning]
Add into in 500ml glass bottle with cap.(do not fully tighten cap onto bottle so steam can vent while heating)
2.5 g
[1x PBS]
?. [Prep brain for sectioning]
Heat in microwave for .Check to see if all agarose is dissolved. If not re-heat at  intervals till completely dissolved.
?. [Prep brain for sectioning]
Pour agarose into and orient brain in mold.
?. [Prep brain for sectioning]
Allow agarose to set about (will change from transparent to slighly cloudy and will become firm to the touch.)
?. [Prep brain for sectioning]
Prep Vibratome with
?. [Prep brain for sectioning]
Adjust vibratome settings:Amplitude 1.00mmSpeed .70mm/sFeed (thickness) 100um
?. [Prep brain for sectioning]
Fill buffer tray with
[1x PBS]
?. [Section brain]
Section desired region at 50um or 100um.
?. [Section brain]
Float sections in (Single section per well) with each well filled with (Label plate and wrap with aluminum foil)
[1x PBS]
?. [DAPI stain]
DAPI stain
?. [DAPI stain]
Add  to
[DAPI stock]
[1xPBS]
?. [DAPI stain]
Select desired sections and remove PBS from wells.
?. [DAPI stain]
Add  of DAPI stain to each well for
[per well]
?. Rinse 2 times  with
[1x PBS]
?. [Mount and coverslip with Vecatshield]
Mount sections on
?. [Mount and coverslip with Vecatshield]
Allow sections to dry for at least
?. [Mount and coverslip with Vecatshield]
Rehydrate slides in  for
[1x PBS]
?. [Mount and coverslip with Vecatshield]
Cover-slip slides with  and allow to fully dry for
[vecatshield]