Mar 25, 2020
Protocol for mouse perfusion with dye, DAPI staining, and slide preparation
Forked from a private protocol
- 1HHMI Janelia Research Campus
Protocol Citation: Boaz Mohar, Monique Copeland 2020. Protocol for mouse perfusion with dye, DAPI staining, and slide preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.59jg94n
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: August 07, 2019
Last Modified: March 25, 2020
Protocol Integer ID: 26635
Materials
MATERIALS
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
Falcon Tube (50 mL)Fischer Scientific
DMSO
Disposable gloves, nitrile
Surflo Winged Infusion Set 21Gx3/4TerumoCatalog #SV*21BLK
Surgical ScissorsFine Science ToolsCatalog #14090-09
Standard Pattern ForcepsFine Science ToolsCatalog #11001-12
ForcepsFine Science ToolsCatalog #11223-20
IsofluranePenn Veterinary Supply, Inc.Catalog #CED1360
STEP MATERIALS
Dimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650
Falcon Tube (50 mL)Fischer Scientific
Isoflurane 1-3%Patterson VeterinaryCatalog #07-893-1389
Silver blue double edge razor blade (Gillette)AmazonCatalog #884718268996
cell culture plate 24 wellSigma-aldrich
Superfrost Plus Microscope SliesThermo Fisher ScientificCatalog #4951PLUS4
PBSFisher ScientificCatalog #BP24384
Agarose LEElectron Microscopy SciencesCatalog #87046
PBSFisher ScientificCatalog #BP24384
DAPIThermo ScientificCatalog #D1306
VECTASHIELD® Hardset™ Antifade Mounting MediumVector LaboratoriesCatalog #H-1400
PBSFisher ScientificCatalog #BP24384
Protocol materials
Falcon Tube (50 mL)Fischer Scientific
In Materials, Materials, Step 5
VECTASHIELD® Hardset™ Antifade Mounting MediumVector LaboratoriesCatalog #H-1400
Materials, Step 14.3
DMSO
Materials
Agarose LEElectron Microscopy SciencesCatalog #87046
Materials, Step 10.1
Silver blue double edge razor blade (Gillette)AmazonCatalog #884718268996
Materials, Step 11
ForcepsFine Science ToolsCatalog #11223-20
Materials
Surflo Winged Infusion Set 21Gx3/4TerumoCatalog #SV*21BLK
Materials
Paraformaldehyde fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)
Materials
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Materials, Step 1
DAPIThermo ScientificCatalog #D1306
Materials, Step 13.1
Disposable gloves, nitrile
Materials
cell culture plate 24 wellMerck MilliporeSigma (Sigma-Aldrich)
Materials, Step 12.1
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
Materials
Standard Pattern ForcepsFine Science ToolsCatalog #11001-12
Materials
Superfrost Plus Microscope SliesThermo Fisher ScientificCatalog #4951PLUS4
Materials, Step 14
PBSFisher ScientificCatalog #BP24384
In Materials, Materials, Materials and 3 steps
Surgical ScissorsFine Science ToolsCatalog #14090-09
Materials
IsofluranePenn Veterinary Supply, Inc.Catalog #CED1360
Materials
Isoflurane 1-3%Patterson VeterinaryCatalog #07-893-1389
Materials, Step 8.1
Prepare dye and fix
Prepare dye and fix
Add 100 µL of DMSO to lyophilized dye (100nmol) and Pipette up and down ~20 times to get 1mM concentration stock.
Dimethyl sulfoxide (DMSO)VWR InternationalCatalog #D2650
Mix further using a vortex machine to ensure the dye is evenly distributed in the DMSO
Add 50-100 ul of 1mM dye stock to 50 mL per mouse of 4% paraformaldehyde in .1M PB
Set-up hood for perfusion
Set-up hood for perfusion
Set up fume hood with:
Equipment
Dissection tray
NAME
VWR
BRAND
470006-956
SKU
Equipment
Peristaltic pump-102R
NAME
Watson-Marlow
BRAND
013.7101.000
SKU
Equipment
Surgical Scissors
NAME
Fine Science tools
BRAND
14090-09
SKU
LINK
Equipment
Forceps
NAME
Fine Science tools
BRAND
11223-20
SKU
LINK
Falcon Tube (50 mL)VWR International
Perfusion and dissection
Perfusion and dissection
Perfusion pump preparation
Attach needle:
Equipment
Surflo winged infusion set 21G 3/4
NAME
Terumo
BRAND
TER 3SV-21BLK
SKU
to pump tubing and flush with ~20 mL of 1x PBS
PBSVWR InternationalCatalog #BP24384
to clear tubing of previous solution
Retreive animal per vivarium protocol
Anesthetize mouse in 2000ml glass beake
pour 5 mL isoflorane down the side of the container.
Isoflurane 1-3%VWR InternationalCatalog #07-893-1389
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)
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.
Begin perfusion
Lay mouse on its back and place a pin in each front paw, others as needed.
Use scissors to cut parallel to the sternum, opening the chest cavity and exposeing the heart.
Insert winged needle into the right ventricle.
Cut left atrium.
Start perfusion pump and flush 50 mL 4% PFA with 100 µL of Dye 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.)
"Stop" perfuison pump when all fixative is flushed through the animal.
Unpin mouse from dissection tray and decapitate behind ears.
(Place carcass in small biohazard bag for disposal.)
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.
Use forceps to peel off skull cap and scope out brain.
Post-fix brain in 20 mL 4% PFA 16:00:00 at 4 °C
Rinse 3 times 00:15:00 00:15:00 00:15:00 in PBS
PBSVWR InternationalCatalog #BP24384
at Room temperature
Prep brain for sectioning
Prep brain for sectioning
Prepare 50 mL 4% agarose
Add 2.5 g
Agarose LEVWR InternationalCatalog #87046
into 50 mL 1x PBS
PBSVWR InternationalCatalog #BP24384
in 500ml glass bottle with cap.
(do not fully tighten cap onto bottle so steam can vent while heating)
Heat in microwave for 00:01:20 .
Check to see if all agarose is dissolved. If not re-heat at 00:00:10 intervals till completely dissolved.
Pour agarose into
Equipment
Peel-A-Way® Embedding Mold (Square - S22)
NAME
polysciences
BRAND
18646A-1
SKU
and orient brain in mold.
Allow agarose to set about 00:10:00
(will change from transparent to slighly cloudy and will become firm to the touch.)
Prep Vibratome
Equipment
VT 1200s Vibratome
NAME
Leica
BRAND
1491200S001
SKU
LINK
with
Silver blue double edge razor blade (Gillette)VWR InternationalCatalog #884718268996
Adjust vibratome settings:
Amplitude 1.00mm
Speed .70mm/s
Feed (thickness) 100um
Fill buffer tray with 300 mL 1x PBS
Section brain
Section brain
Section desired region at 50um or 100um.
Float sections in
cell culture plate 24 wellVWR International
(Single section per well) with each well filled with 500 µL 1x PBS
(Label plate and wrap with aluminum foil)
DAPI stain
DAPI stain
DAPI stain
Add 2.1 µL DAPI stock to 50 mL 1xPBS
DAPIVWR InternationalCatalog #D1306
Select desired sections and remove PBS from wells.
Add 500 µL per well of DAPI stain to each well for 02:00:00
Rinse 2 times 00:05:00 00:05:00 with 500 µL 1x PBS
Mount and coverslip with Vecatshield
Mount and coverslip with Vecatshield
Mount sections on
Superfrost Plus Microscope SliesVWR InternationalCatalog #4951PLUS4
Allow sections to dry for at least 02:00:00
Rehydrate slides in 250 mL 1x PBS for 00:30:00
Cover-slip slides with 130 µL vecatshield and allow to fully dry for 12:00:00
VECTASHIELD® Hardset™ Antifade Mounting MediumVWR InternationalCatalog #H-1400