morphoFISH Protocols

MouseLight Project Team; Tiago A Ferreira

Mar 13, 2025

morphoFISH Protocols

DOI

dx.doi.org/10.17504/protocols.io.q26g7pej3gwz/v1

MouseLight Project Team¹,
Tiago A Ferreira¹

¹HHMI - Janelia Research Campus

Monique Copeland

HHMI Janelia Research Campus

DOI: dx.doi.org/10.17504/protocols.io.q26g7pej3gwz/v1

Protocol Citation: MouseLight Project Team, Tiago A 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 08, 2024

Last Modified: March 13, 2025

Protocol Integer ID: 94907

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

ReagentNuclease-free water or water filtered using a Milli-Q filtering systemAmbionCatalog #AM9932  ReagentParaformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714  
Reagent4-Hydroxy-TEMPOMerck MilliporeSigma (Sigma-Aldrich)Catalog #176141  ReagentN,N,N′,N′-TetramethylethylenediamineMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7024  ReagentAmmonium persulfateContributed by usersCatalog #A3678  ReagentDMSO, AnhydrousThermo FisherCatalog #D12345  
ReagentMethacrylic acid N-hydroxysuccinimide esterMerck MilliporeSigma (Sigma-Aldrich)Catalog #730300-1G  Reagent40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140  ReagentOptiPrep - Density Gradient Media (Iodixanol)Cosmo BioCatalog #AXS-1114542  ReagentAcrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9099  ReagentN-N-Bis(acryloyl)Cystamine Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4929-5G  Reagent10x Phosphate Buffered Saline Gibco, ThermoFisherCatalog #70011044  ReagentAgarose-Ultra PureInvitrogen - Thermo FisherCatalog #16500-500  ReagentRNAse AWAYMolecular BioProductsCatalog #7000  ReagentMOPS pH 7Thermo Fisher ScientificCatalog #AAJ61821AK  
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301  
ReagentPeel Away Disposable Embedding MoldsElectron Microscopy SciencesCatalog #70183  


Recipes: 
Reagents: 
 
ABCDE
4HT0.5 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
  
ABCDEF
   
    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
 
ABCDE
  Melphalan
    2mg/mL
    250mg
    125mL DMSO
    store -20°C for
  several months
  
**Make solution on a hot plate with a stir bar.  Mix for Duration02:00:00   at Temperature37 °C     until completely dissolved.  Aliquot and store at Temperature-20 °C   for two months.  Discard if turns yellow in color.


Perfusion buffers:
ABCDE
Perfusion fixative    
   tot vol: 50 mL
 Stock  final volume (mL)
32% PFA (%) 32 4 6.25 
Nuclease free water   43.75 
 
ABCDE
Protein anchoring solution    
   tot vol: 50 
 Stock Final Volume 
PFA32 4 6.25 
Nuclease free water42.7
MA-NHS (mM) 1000 20 1 
 **Add MA-NHS immediately prior to perfusion**

ABCDE
         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 (%)10.21  
4HT (5) .50.011  
APS (ppt)10.21  
water         10.5
 ***Add 4HT, TEMED, and APS immediately prior to perfusion

  
ABCDE
  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
  
 

Brain Perfusion and Fixation

Protocol
NAME
morphoFISH Perfusion
CREATED BY
Monique Copeland

Aqueous Delipidation

Protocol
NAME
Aqueous (SBiP) Delipidation for Whole Mouse Brain After morphoFISH Perfusion 
CREATED BY
Monique Copeland

Agarose Embedding (5%Agarose)

1m 30s

Rinse 500ml glass bottle with RNAse AWAY.

Add Amount100 mL   1x PBS to beaker and warm in microwave for Duration00:01:30  
Note
Use caution glass bottle will be hot. Use appropriate protection.

1m 30s

Add Amount5 g   agarose powder to warm 1x PBS, mix well by rotating the bottle.

Place glass bottle in Temperature57 °C   incubator for Duration02:00:00   or until agarose is dissolved 

Submerge the brain in Amount25 mL agarose  in a 50mL conical tube for Duration00:15:00   Temperature37 °C   

Note
Equilibrating the brain to agarose helps create a better seal around the brain when embedding in the mold.  Store remaining agarose at Temperature37 °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 Duration00:10:00   Temperature4 °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

Equilibrate brain in Amount100 mL  30% DMSO for Duration72:00:00   aShaker40 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.  

Equilibrate brain in Amount100 mL   50:50 DMSO:Optiprep forDuration72:00:00  atShaker40 rpm, Room temperature  protected from light 

Equilibrate brain in Amount100 mL   60:40 DMSO:Optiprep for Duration72:00:00   at Shaker40 rpm, Room temperature   protected from light.

Replace with Amount100 mL   60:40 DMSO:Optiprep for Duration168:00:00  (7-10 days or until brain sinks to bottom) at Shaker40 rpm, Room temperature   protected from light.

Tissue Sectioning and imaging

10m

Trim agarose block as needed

Glue block to specimen disc and fill imaging tray with 60:40 DMSO:Optiprep

Proceed to imaging as per  Economo et al., 2016; Winnubst et al., 2018

Neuronal Reconstruction

Protocol
NAME
Reconstructing Complete Neurons in Whole Brains Using HortaCloud
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 Amount1 mL   RNAse-free water for Duration00:05:00   at TemperatureRoom 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

Rinse 2x with Amount2 mL   70% EtOH for Duration00:05:00   at TemperatureRoom 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.

Store in 70% EtOH at Temperature4 °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
NAME
Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) for morphoFISH
CREATED BY
Mark Eddison

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

	A	B	C	D	E
	Melphalan	2mg/mL	250mg	125mL DMSO	store -20°C for several months

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 (5)	.5	0.01		1
APS (ppt)	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

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morphoFISH Protocols