Tissue Clearing:
- iDISCO (Renier Lab)
- 3DISCO (Chedotal Lab)
- Our 3DISCO Clearing-protocol-Version_Dec_2019 (adapted from protocol Alain Chedotal-Morgane Belle)
- 3DISCO Star Protocol (including extensive troubleshooting, imaging tips and tricks, Imaris based analysis)
Microscope:
- Tips and tricks for imaging with lightsheet microscope; Ultramicroscope II (See section: Ultramicroscopy imaging)
- Ultramicroscope II – Imaging settings table
Imaris analysis:
Validated antibodies:
| Antigen | Host | Vendor | Catalog | Dilution | Type of staining |
| Calbindin | Rabbit | Swant | CB -38a | 1:15000 | 3DISCO |
| Cfos | Rabbit | Synaptic Systems | 226003 | 1:500 | iDISCO |
| CHX10 (VSX2) |
Sheep | Abcam | ab16141 | 1:500 | 3DISCO |
| CTIP2 | Rat | Abcam | ab18465 | 1:100 | iDISCO |
| GFP | Rabbit | Invitrogen | A11122 | 1:1000 | 3DISCO |
| Islet1 | Goat | Neuromics | GT15051 | 1:2000 | 3DISCO |
| L1 | Rat | Milipore | MAB5272 | 1:500 | 3DISCO |
| Netrin | Rabbit | Abcam | ab126729 | 1:500 | 3DISCO |
| Nrp2 | Goat | R&D | AF567 | 1:1000 | 3DISCO |
| Pax6 | mouse | DSHB | pax6 | 1:200 | iDISCO |
| pHH3 | Rabbit | Cell Signaling | 9701 | 1:800 | 3DISCO |
| RFP | Rabbit | Rockland | 600-401-379 | 1:1000 | iDISCO |
| Robo1 | Goat | R&D | AF1749 | 1:300 | 3DISCO |
| Robo3 | Goat | R&D | AF3155 | 1:300 | 3DISCO |
| Satb2 | Rabbit | Abcam | ab34735 | 1:200 | iDISCO |
| Six3 | Rabbit | Rockland | 600-401-A26 | 1:500 | 3DISCO |
| TH | Sheep | Pelfreez | P60101-150 | 1:500 | 3DISCO |
| TH | Rabbit | Millipore | AB152 | 1:1000 | 3DISCO / iDISCO |
| TrkA | Goat | R&D | AF1056 | 1:400 | 3DISCO |
| TrkC | Goat | R&D | AF1404 | 1:400 | 3DISCO |
Considerations for the choice of secondary antibodies:
|
Laser wavelength |
Suggested fluorophore |
Advantages |
Disadvantages |
|
488 nm |
Alexa Fluor 488, Cy2 |
Compatible with the 561 and 647 nm laser. |
Highest autofluorescence. Least preferred channel for imaging. |
|
561 nm |
Alexa Fluor 555, Alexa Fluor 568, Cy3, Cy3.5 |
Strong laser power; Compatible with 488 and 647 nm lasers. |
Reasonable amount of autofluorescence. |
|
647 nm |
Alexa Fluor 647, Cy5 |
Very low autofluorescence; strong light penetration; strong laser power; Compatible with 561 and 488 nm lasers. |
Most recommended channel for imaging. |
|
730 nm |
Alexa Fluor 750, Cy7 |
Almost no autofluorescence; strong light penetration. |
Weak laser power |
Note: The 730 nm laser is positioned in a separate beam combiner which might cause minor alignment variations in the light sheet angles. Pixel-based co-localization analysis should not be performed with this laser.