Mar 14, 2025
morphoFISH Protocols
- MouseLight Project Team1,
- Tiago Ferreira1
- 1HHMI - Janelia Research Campus
Protocol Citation: MouseLight Project Team, Tiago Ferreira 2025. morphoFISH Protocols . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pej3gwz/v1
Manuscript citation:
Ferreira AT et al. (2024) Discovery of neuronal cell types by pairing whole cell reconstructions with RNA expression profiles. bioRxiv 2024.12.30.630829, https://doi.org/10.1101/2024.12.30.630829
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: February 09, 2024
Last Modified: March 14, 2025
Protocol Integer ID: 94907
Keywords: reconstructed neuron, assisted iterative fluorescence in situ hybridization, brain imaging, neurons in the mouse brain, tissue clearing, mouse brain, assisted iterative fluorescence, imaging, neuron, situ hybridization
Funders Acknowledgements:
HHMI
Grant ID: N/A
Abstract
morphoFISH combines tissue clearing, submicron whole-brain imaging, and Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to assign molecular identities to fully reconstructed neurons in the mouse brain. This protocol aggregates the morphoFISH procedures, including perfusion, tissue-clearing, and probing of transcripts.
Image Attribution
Tiago Ferreira
Materials
Nuclease-free water or water filtered using a Milli-Q filtering systemAmbionCatalog #AM9932 Paraformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714
4-Hydroxy-TEMPOMerck MilliporeSigma (Sigma-Aldrich)Catalog #176141 N,N,N′,N′-TetramethylethylenediamineMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7024 Ammonium persulfateCatalog #A3678 DMSO, AnhydrousThermo FisherCatalog #D12345
Methacrylic acid N-hydroxysuccinimide esterMerck MilliporeSigma (Sigma-Aldrich)Catalog #730300-1G 40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140 OptiPrep - Density Gradient Media (Iodixanol)Cosmo BioCatalog #AXS-1114542 AcrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9099 N-N-Bis(acryloyl)Cystamine Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4929-5G 10x Phosphate Buffered Saline Gibco - Thermo Fisher ScientificCatalog #70011044 Agarose-Ultra PureInvitrogen - Thermo FisherCatalog #16500-500 RNAse AWAYMolecular BioProductsCatalog #7000 MOPS pH 7Thermo Fisher ScientificCatalog #AAJ61821AK
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Peel Away Disposable Embedding MoldsElectron Microscopy SciencesCatalog #70183
Recipes:
Reagents:
| 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 | |
| A | B | C | D | E | ||
| 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 |
Melphalan
| A | B | C | D | E | |
| Melphalan | 2mg/mL | 250mg | 125mL DMSO | store -20°C for several months |
**Make solution on a hot plate with a stir bar. Mix for 02:00:00 at 37 °C until completely dissolved. Aliquot and store at -20 °C for two months. Discard if turns yellow in color.
Perfusion buffers:
| 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 |
**Add MA-NHS immediately prior to perfusion**
| 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 (5) | .5 | 0.01 | 1 | ||
| APS (ppt) | 10 | .2 | 1 | ||
| water | 10.5 |
***Add 4HT, TEMED, and APS immediately prior to perfusion
| 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 |
Troubleshooting
Brain Perfusion and Fixation
Aqueous Delipidation
Protocol
CREATED BY
Monique Copeland
Agarose Embedding (5%Agarose)
1m 30s
Rinse 500ml glass bottle with RNAse AWAY.
Add 100 mL 1x PBS to beaker and warm in microwave for 00:01:30
Note
Use caution glass bottle will be hot. Use appropriate protection.
1m 30s
Add 5 g agarose powder to warm 1x PBS, mix well by rotating the bottle.
Place glass bottle in 57 °C incubator for 02:00:00 or until agarose is dissolved
2h
Submerge the brain in 25 mL agarose in a 50mL conical tube for 00:15:00 37 °C
Note
Equilibrating the brain to agarose helps create a better seal around the brain when embedding in the mold. Store remaining agarose at 37 °C for later use.
15m
Prepare 20x20 embedding mold by inserting two needles parallel to each other through the side of the mold about 2-3mm from the bottom. This creates a platform for the brain to rest while agarose is setting, ensuring brain remains in the middle of the mold.
Fill embedding mold with 15mL of 5% agarose
Quickly place the brain in the center of the mold resting on the needles and allow agarose to set 00:10:00 4 °C
Note
The agarose will turn slightly white in color and harden to the touch when it is completely set.
10m
Remove the agarose block from the mold by gently pulling the edges of the mold apart. Trim the block as desired and proceed immediately to index matching.
Index Matching
6d
Equilibrate brain in 100 mL 30% DMSO for 72:00:00 a40 rpm, Room temperature protected from light.
Note
The agarose block will initially float in each index matching solution, but will gradually sink as it equilibrates to the solution.
3d
Equilibrate brain in 100 mL 50:50 DMSO:Optiprep for72:00:00 at40 rpm, Room temperature protected from light
3d
Equilibrate brain in 100 mL 60:40 DMSO:Optiprep for 72:00:00 at 40 rpm, Room temperature protected from light.
3d
Replace with 100 mL 60:40 DMSO:Optiprep for 168:00:00 (7-10 days or until brain sinks to bottom) at 40 rpm, Room temperature protected from light.
1w
Tissue Sectioning and imaging
10m
Trim agarose block as needed
Glue block to specimen disc and fill imaging tray with 60:40 DMSO:Optiprep
Neuronal Reconstruction
Protocol
CREATED BY
Tiago A Ferreira
Slice collection
10m
Collect slice from imaging tray with sterile rod or brush into a 24-well plate. Briefly shining light into the imaging bath may assist in locating the brain slices for recovery
Rinse with 1 mL RNAse-free water for 00:05:00 at Room temperature to rinse off the DMSO. Trace amounts of DMSO left in the tissue may impact RNA transcript detection by hybridization chain reaction (HCR). Slices become opaque in aqueous solutions
5m
Rinse 2x with 2 mL 70% EtOH for 00:05:00 at Room temperature
Note
XFP labeling (GFP, tdTomato, etc.) will become dim in 70% EtOH. Signal strength should recover once the brain slice is rehydrated in 1x PBS.
5m
Store in 70% EtOH at 4 °C until further processing. Seal well plate with parafilm (or sealing film) to minimize evaporation. Brain slices are stable for at least 3 months, although it is recommended to proceed to RNA detection as quickly as possible
Note
Check ethanol level in the wells frequently to monitor evaporation. Replace 70% EtOH as needed.
RNA detection via EASI-FISH
20m
Protocol
CREATED BY
Mark Eddison
6d