Mar 13, 2025
morphoFISH Perfusion
Forked from a private protocol
- 1Scientific Operations, Janelia Research Campus;
- 2HHMI Janelia Research Campus;
- 3Allen Institute for Neural Dynamics
Protocol Citation: Monique Copeland, Paul Tillberg, Jayaram Chandrashekar, Tiago A Ferreira 2025. morphoFISH Perfusion. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8d67v5b/v1
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: September 30, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 108683
Funders Acknowledgements:
Howard Hughes Medical Institute
Grant ID: N/A
Abstract
morphoFISH perfusion is a fixation protocol aimed at maximal retention of RNA in the mby crosslinking RNA and proteins to the scafold formed by fixatives and an embedding hidrogel. As a result of the high retention, mRNA transcripts can be detected after long histological procedures such as tissue clearing.
Materials
MATERIALS
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
(PBS)
Falcon Tube (50 mL)Fischer Scientific Paraformaldehyde (prilled 95%)Merck MilliporeSigma (Sigma-Aldrich)Catalog #441244
N,N,N′,N′-TetramethylethylenediamineMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7024
(TEMED)
4-Hydroxy-TEMPOMerck MilliporeSigma (Sigma-Aldrich)Catalog #176141
(4HT)
Nuclease-free water or water filtered using a Milli-Q filtering systemAmbionCatalog #AM9932 MOPS pH 7Thermo Fisher ScientificCatalog #AAJ61821AK N-N-Bis(acryloyl)Cystamine Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4929-5G
(BAC)
40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140 Methacrylic acid N-hydroxysuccinimide esterMerck MilliporeSigma (Sigma-Aldrich)Catalog #730300-1G
(MA-NHS)
DMSO, AnhydrousThermo FisherCatalog #D12345 Ammonium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3678
(APS)
RNase AWAY™ Surface DecontaminantCarl RothCatalog #A998.4
Equipment
Standard Pattern Forceps
NAME
Tools
TYPE
Fine Science Tools
BRAND
11001-12
SKU
LINK
IsofluranePenn Veterinary Supply, Inc.Catalog #CED1360
Equipment
Surflo winged infusion set 21G 3/4
NAME
Terumo
BRAND
TER 3SV-21BLK
SKU
Equipment
Surgical Scissors
NAME
Fine Science tools
BRAND
14090-09
SKU
LINK
Equipment
Forceps
NAME
Fine Science tools
BRAND
11223-20
SKU
LINK
Equipment
Tygon E-1000 Tubing, 1/8" ID x 1/4" OD; 50 Ft
NAME
tubing
TYPE
Tygon
BRAND
EW-50106-11
SKU
LINK
Equipment
Watson-Marlow 120S Variable-Speed Single-Channel Pump System
NAME
Peristaltic pump
TYPE
Watson Marlow
BRAND
EW-50130-00
SKU
LINK
Recipes
| A | B | C | D | E | |
| 4HT | 0.5 wt% | 50mg | 9.95mL water | store -20°C, several months | |
| TEMED | 10 vol% | 50uL | 450uL water | store single use aliquotes at -20°C, several months | |
| APS | 10 wt% | 50mg | 450uL water | store single use aliquotes at -20°C, several months |
| A | B | C | D | E | F | |
| 1 | 1M MA-NHS | 1M | 186.3mg | 1mL anhydrous DMSO | Make fresh day of perfusion | |
| 2 | BAC | 20% | 120mg | 600ul anhydrous DMSO | Make fresh day of perfusion |
| A | B | C | D | E | |
| Melphalan | 2mg/mL | 250mg | 125mL anhydrous DMSO | store single use aliquots at -20°C |
Make solution on a hot plate with a stir bar. Mix for 02:00:00 or until dissolved at 37 °C until completely dissolved. Aliquot (single use aliquots, i.e., 50mL/perfusion) and store at -20 °C for two months. Discard if turns yellow in color.
Note
Melphalan is highly reactive. Variability between lots is expected if e.g., the container overheated or was mishandled during delivery. Being a nitrogen mustard, it can react with itself, water, etc. To minimize such variability, dissolve exclusively in anhydrous DMSO, and monitor color changes in the solution. Open container only once and use only single-use frozen aliquots.
| A | B | C | D | E | |
| Perfusion fixative | |||||
| tot vol: | 50 mL | ||||
| Stock | final | volume (mL) | |||
| 32% PFA (%) | 32 | 4 | 6.25 | ||
| Nuclease free water | 43.75 |
| A | B | C | D | E | |
| Protein anchoring solution | |||||
| tot vol: | 50 | ||||
| Stock | Final | Volume | |||
| PFA (%) | 32 | 4 | 6.25 | ||
| Nuclease free water | 42.7 | ||||
| MA-NHS (mM) | 1000 | 20 | 1 |
| A | B | C | D | E | |
| tot vol: | 50 | ||||
| Gel solution | |||||
| stock | final | volume | |||
| AcAm (%) | 40 | 20 | 25 | ||
| BAC (%, in DMSO) | 20 | 0.1 | 0.25 | ||
| PB pH7.5 (mM) | 1000 | 100 | 5 | ||
| PFA (%) | 32 | 4 | 6.25 | ||
| TEMED pH7.5 (%) | 10 | .2 | 1 | ||
| 4HT (wt%) | .5 | 0.01 | 1 | ||
| APS (wt%) | 10 | .2 | 1 | ||
| water | 10.5 |
| A | B | C | D | E | |
| A | B | C | D | ||
| Post-fixation solution | |||||
| Tot vol: | 50 | ||||
| Stock | Final | Volume (mL) | |||
| PFA (%) | 32 | 4 | 6.25 | ||
| Glutaraldehyde (%) | 10 | .05 | .250 | ||
| MOPS pH 7 (mM) | 1000 | 10mM | 1 | ||
| Melphalan (mg/mL) | 2 | 1 | 25 | ||
| Nuclease free water | 17.5 |
Prepare solutions and set up fume hood for perfusion
Prepare solutions and set up fume hood for perfusion
Thaw aliquoted reagents to room temperature and prepare perfusion buffers: perfusion fixative, protein anchoring solution, gel solution.
1h
Prepare 50 mL beaker 1/3 filled with 5 mm glass beads. Rinse both with RNase AWAY‱ prior to use.
Set up fume hood with dissection tray, perfusion pump, 4 50 mL conical tubes containing the perfusion solutions, forceps, scissors, and biohazard bag. Clean all tools with RNase AWAY‱ prior to use.
15m
Sequential Perfusion
Sequential Perfusion
1d 17h 30m
1d 17h 30m
Prepare perfusion pump by attaching butterfly needle to the tubing. Start the pump and flush aprox. 20 mL of 1x RNAse-free PBS to wash tubing. Stop the pump. Make sure no bubbles are in the line.
Retrieve animal per vivarium protocol.
Prepare anesthesia chamber by pouring aprox. 5 mL isoflurane into the bottom of desiccation jar and placing a paper towel on the porcelain insert.
1m
Place the animal inside the container on the insert and cover.
Note
Do not allow liquid isoflurane to come into contact with the animals’ skin as this can cause irritation.
3m
Monitor breathing. Once the mouse respiration reduces to 2 breaths per second, check for plantar reflex by gently pinching the foot with forceps. If no reaction, remove the mouse from the jar and place on dissection tray. If the mouse reacts close the lid and wait ~30seconds. Check reflex again until no reflex observed.
Note
Timing is extremely important. Over-exposure to isoflurane can lead to cardiac arrest. Premature cell death and blood clots will impact the quality of the perfusion
Begin perfusion.
Note
When transitioning between perfusion solutions make sure to stop the pump prior to moving intake tubing to prevent bubbles from forming. Perfusing air bubbles can cause major deformations, including in brain ventricles.
Lay mouse on its back and place a pin in each front paw, others as needed.
Carefully use scissors to open the chest cavity by lifting the base of the sternum and cutting parallel to either side of the sternum to the neck, then cut the diaphragm without puncturing the heart or lungs. Expose the heart.
Use forceps to gently hold the heart by the right ventricle. Insert the tip of the butterfly needle into the bottom apex of the left ventricle. Cut the right atrium to allow for drainage
NB:
- (Optional) Use a 18G needle to secure the butterfly needle in place or a hemostat clamp.
- Correct placement of the needle is important. Do not push the needle too deep. Only insert the tip of the needle just past the beveled angle of the tip.
Start perfusion pump and flush 10 mL 1xPBS at a rate of 7.5mL/min.
Note
Liver should turn pale tan color during. If the liver does not become lighter, check needle position to ensure it did not puncture through the ventricle wall.
Stop perfusion pump. Transfer intake tubing to the conical tube with the first perfusion solution, i.e., 4 % (v/v) PFA . Start the pump and flush 50 mL
NB:
- Muscle contractions are poised to occur. If not, check placement of the needle and reposition as needed
- Intestines and liver may extend outside of the abdominal cavity and displace needle
10m
When only 10 mL of fixative remains, add 1 mL 1M MA-NHS to anchoring solution. Mix well by inverting 5x. Stop perfusion pump and transfer intake tubing to anchoring solution freshly supplemented with MA-NHS. Start the pump and flush 50 mL of protein anchoring solution
10m
When only 10 mL of protein anchoring solution remains, add 1 mL 10% TEMED , 1 mL 0.5% 4HT , and1 mL 10% APS to gel solution. Mix well by inverting x5. Stop perfusion pump and transfer tubing to gel solution. Start the pump and flush 50 mL gel solution
10m
Stop the pump and remove the needle from the ventricle. Flush the tubing with 20 mL PBS to purge gel solution.
Remove animal from dissection tray.
Dissect out the brain.
Polymerize perfused gel by placing the brain in the beaker with glass beads (ventral side down). Place beaker in sealable plastic bag.
Remove oxygen from the bag by filling the bag with Nitrogen gas from a compressed tank. Replace bag's atmosphere a second time by compressing bag and refilling with Nitrogen gas.
NB: The Nitrogen source should be near the dissection table so that the gel can polymerize as quickly as possible.
Seal bag and gel for 01:00:00 at Room temperature
1h
Prepare 50 mL of post-fixation solution. Transfer brain to post-fixation solution for 40:00:00 at 40 rpm, 4°C
1d 16h
Rinse 3x with RNAse free PBS for00:30:00 , at 40 rpm, Room temperature
Brain can be stored in 1x PBS @ 4C for further processing. For optimal detection of RNA it is recommended to avoid delays to prevent RNA degradation.
30m