Multiple retrograde tracing methods compatible with 3DISCO clearing

Figures & data

Figure 1. Target muscles before and after intramuscular injections of tracers. (A) Before injection. (B) After injection: target muscles were completely Coloured.

Figure 2. Harvest of the spinal cord (spinal nerve roots remained).

Table 1. Properties of the retrograde tracers and setups of lasers and filters of Zeiss LSM 780 confocal microscope.

Tracers	Solvent	Concentration (wt/vol) (%)	Ex peak (nm)	Em peak (nm)	Lasers (nm)	Filters (nm)
FG	Distilled water	5	330/390*	450/600*	405	410–499
CTb-488	1 × PBS	10	495	519	488	491–552
FR	Saline	10	555	580	561	552–638
CTb-647	1 × PBS	10	650	668	633	638–755

FG: Fluoro Gold; CTb-488: Cholera Toxin Subunit B (Recombinant), Alexa Fluor™ 488 Conjugate; FR: Fluoro Ruby; CTb-647: Cholera Toxin Subunit B (Recombinant), Alexa Fluor™ 647 Conjugate.

* FG have two excitation peaks at approx. 330 and 390 nm, and two emission peaks at approx. 450 and 600 nm. The values of E_x and E_m peak are variable in different PH of FG solution.

Figure 3. Spinal cord before and after optical clearing procedure. (A) Before clearing. (B) After clearing.

Figure 4. MIP images of single track. (A) Tibialis anterior motor neurons labelled by FG. (B) Soleus motor neurons labelled by CTb-488. (C) Gastrocnemius motor neurons labelled by CTb-488. (D) Lateral muscle motor neurons labelled by CTb-647.

Figure 5. MIP images of multi-tracks in a holistic view [motor neurons innervating tibialis anterior (blue), gastrocnemius (red), soleus (green), lateral muscle (magenta)]. White arrows: dendrites or axons around the soma.

Table 2. Imaging results of motor neuron pools labelled by the four tracers.

Tracers	Lasers power (at 10× magnification) (%)	Signal intensity	SNR	Pseudo-colours
FG	15	++	+	Blue
FR	10	+++	++	Red
CTb-488	10	+++	++	Green
CTb-647	4	+++	+++	Magenta

SNR: signal to noise ratio.

Figure 6. Three dimensional reconstruction of the labelled neurons. (A) Snapshot of the 3D reconstruction. (B) Vantage analysis of the four motor neuron pools.