TITLE: mFISH3D
AUTHORS: Tatsuya Murakami

[DESCRIPTION]
The protocol is for the multiplexed in situ hybridization in 3D (mFISH3D) in an adult mouse brain / a block of a human brain. 

[BEFORE_START]
If you worry about the contamination of RNAse, use RNAse-free water to make all the reagents.

[GUIDELINES]
See safety data sheets for proper chemical handling, precautionary measures, and waste disposal.
Obey all local regulations/guidelines for handling and disposal of used reagents and solutions containing reagents mixed in.

[STEPS]
SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
1. Incubate tissue in 4% paraformaldehyde (pH 6.5~7.0) overnight at 4°C. 

  
 


SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
2. (optional) Subdissect the tissue. The dissected volume will be the final volume. 



SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
3. For the following sample preparation steps, use 4 ml of solution per one tissue block.
I recommend using a 5 ml Eppentube or 15 ml Falcon tube.

Wash the tissue in 2xSSC, 4 mM citric acid. Gently shake at 4°C.
30 min 
Refresh 2xSSC 4 mM citric acid solution. Gently shake at 4°C.
30 min 

 

SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
4. Start dehydration by immersing the tissue in gradient concentrations of methanol (MeOH).
Replace the solution with 30% MeOH. Gently shake at RT.
30 min 

Replace the solution with 50% MeOH. Gently shake at RT.
30 min 

Replace the solution with 80% MeOH. Gently shake at RT.
30 min 

Replace the solution with 100% MeOH. Gently shake at RT.
30 min 

Refresh 100% MeOH. Gently shake at RT.
30 min 

Refresh 100% MeOH. Move the tissue to 37°C and keep gentle shakes.
Shake the tissue for 16-48 hrs depending on the tissue size.
1440 min 
SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
5. Refresh 100% MeOH.
For long-term storage, move the tissue to -20°C. I confirmed RNA is stable for at least 6 months.
SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
6. (optional) The following few steps are optional. 
This step is for photobleaching to quench autofluorescence.
The photobleacihng is critical for human brain imaging while you can skip this step for rodent brains.

Replace the solution with BABB. Gently shake at RT until the tissue gets cleared.
180 min  
If the transparency is not high, refresh BABB and gently shake the tissue for additional hours. 
Move the tissue to a glass cuvette or polypropylene plastic container.

 

SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
7. Photobleach the tissue with a bright LED.
I use the LED from Thorlabs (MWWHLP1) with an adjustable collimation adapter (SM1U25-A) and give max-power illumination from a 2-cm distance.
Illuminate the tissue in BABB for one or two nights until you do not see autofluorescence.
2880 min 
 
 

SECTION: Preprocessing
9. Unless noted, use a 5 ml Eppentube for tissue processing.


Equilibrate the brain in refreshed 100% MeOH at RT.
30 min 
Replace the solution with 80% MeOH. Gently shake at RT.
30 min 
Replace the solution with 50% MeOH. Gently shake at RT.
30 min 
Replace the solution with 30% MeOH. Gently shake at RT.
30 min 
Replace the solution with 4 ml of 10 mM HCl. Gently shake at 37°C.
30 min 
Refresh 10 mM HCl. Gently shake at 37°C overnight.
960 min 

SECTION: Tissue sample preparation:  a whole-mouse brain, blocks of a fresh frozen human brain
8. Wash out BABB by immersing the tissue in 100% MeOH.

Gently shake the tissue at RT.
30 min 

Refresh 100% MeOH. Gently shake at RT.
30 min 

Refresh 100% MeOH. Gently shake at RT.
30 min 

Refresh 100% MeOH.
For long-term storage, move the tissue to -20°C. I confirmed RNA is stable for at least 6 months.
SECTION: Preprocessing
10. 
Rinse the sample with wash buffer 1 (5xSSC, 20 mM citric acid, 0.01% Tween-20) briefly.
Wash the tissue with wash buffer 1 at RT with a gentle shake.
30 min 
Replace the solution with 2 ml of 10 ug/ml proteinase K in washing buffer 2 (2xSSC, 4 mM citric acid, 0.01% Tween-20).  
Shake the tissue at RT for 2 - 5 hours depending on the tissue size.
120 min 
Wash the sample with wash buffer 1 at RT with a gentle shake.
30 min 
Refresh wash buffer 1. Gently shake at RT.
30 min 

SECTION: Primary probe hybridization
11. Use a 2 ml Eppentube to save the oligo probes.
If the sample does not fit a 2ml tube, use a 5 ml tube with double the amount of the solution. 

Move the tissue to 500 ul of hybridization buffer (30% formamide, 5xSSC, 9 mM citric acid, (optionally 3% PEG8000)) for pre-hybridization. Shake gently at RT.
30 min 
Replace the solution with 500 ul of hybridization buffer with 1 micromolar (µM) primary probes. Shake gently at 37°C.
I confirmed the hybridization in a half hemisphere brain can be done in 3 hrs. For a whole mouse brain, I recommend longer incubation up to 16 hrs. 
180 min  or 960 min 


 

SECTION: Primary probe hybridization
12. Wash the tissue with wash buffer 1.  Wash three times in total, 2 hours for each.
120 min 
120 min 
120 min 


 

SECTION: HCR probe hybridization
13. Equilibrate the tissue with 500 ul of hybridization buffer for pre-hybridization. Shake gently at RT.
30 min 

SECTION: HCR probe hybridization
14. During the pre-hybridization, denature HCR probes as follow.
Prepare necessary amounts of HCR probes in PCR microtubes.
Heat the PCR tubes at 95°C for a minute using a thermal cycler.
Remove the tubes from the cycler, and cool down the tubes at RT for ~30 minutes in a dark space.
30 min 

 

SECTION: HCR probe hybridization
15. Replace the solution with 500 ul of hybridization buffer with 90 nM of HCR probes.
Shake gently at RT for more than 16 hours. 
For a whole-mouse brain, incubation of more than 30 hours is recommended.
1800 min 

SECTION: Tissue clearing
16. Wash the tissue with wash buffer 1.  Wash twice in total. 1 hour for each.
60 min 
60 min 
For desalting, replace wash buffer1 with wash buffer 2. Wash twice in total. 1 hour for each.
60 min 
60 min 

 

SECTION: Tissue clearing
17. For dehydration, you can either use a 2 ml tube or a 5 ml tube.

Replace the solution with 30% MeOH. Gently shake at RT.
30 min 
Replace the solution with 50% MeOH. Gently shake at RT.
30 min 
Replace the solution with 80% MeOH. Gently shake at RT.
30 min 
Replace the solution with 100% MeOH. Gently shake at RT.
30 min 
Refresh 100% MeOH. Gently shake at RT.
30 min 

Replace the solution with BABB. Gently shake at RT.
180 min  
Refresh BABB and gently shake the tissue for additional hours until the tissue gets cleared. 
180 min 

Congratulations! The tissue is now ready to be imaged.
Please follow the next step if you want additional cycles.
SECTION: Dehybridization/Photobleaching
18. If you designed the experiment in a way that all the primary probes from different cycles do not have cross-talk, photobleaching is more recommended than dehybridization.
If not, you have to remove the primary probes from the previous cycle.

If you choose to photobleach, check step 7 and ignore the dehybridization step 21.
  

SECTION: Dehybridization/Photobleaching
19. Wash out BABB by immersing the tissue in 100% MeOH.

Gently shake the tissue at RT.
30 min 

Refresh 100% MeOH. Gently shake at RT.
30 min 

Refresh 100% MeOH. Gently shake at RT.
30 min 
(Optional pausing point) You can keep washing in MeOH overnight. Store the sample at -20°C for a long term.
SECTION: Dehybridization/Photobleaching
20. Replace the solution with 80% MeOH. Gently shake at RT.
30 min 
Replace the solution with 50% MeOH. Gently shake at RT.
30 min 
Replace the solution with 30% MeOH. Gently shake at RT.
30 min 
Replace the solution with wash buffer 1. Gently shake at RT.
30 min 
Move the tissue to 500 ul of hybridization buffer for pre-hybridization. Shake gently at RT.
30 min 
SECTION: Dehybridization/Photobleaching
21. Replace the solution with 500 ul of hybridization buffer with 1 μM primary probes.
Move the tissue to 37°C. Gently shake the tissue.
60 min 
Move the tissue to 65°C. Incubate the tissue for 1 hour.
60 min 

SECTION: Dehybridization/Photobleaching
22. Refresh the solution with 500 ul of hybridization buffer with 1 μM primary probes.
Move the tissue to 37°C. Gently shake the tissue for the duration shown in step 11.

Follow the left HCR and clearing steps as described above.