Alexa fluor 488 conjugated dextran

For a subset of experiments, bulk anterograde labeling of regenerated axons was performed in transected (n = 14) and re-transected (n = 7) spinal cords, as previously described (Lau et al., 2013). Briefly, axons were labeled with a fluorescent dye (5 mM Alexa Fluor 488-conjugated dextran; 10 kDa; Thermo Fisher, Inc. Waltham, MA), diluted in lamprey internal solution (180 mM KCl, 10 mM HEPES, pH 7.4) via a 1x1x1 mm piece of Gelfoam (Pfizer; New York, NY), which was applied 5 mm rostral to the lesion site. After application, the dye was allowed to transport for 3–6 days before harvesting the spinal cords. Labeled spinal cords were imaged live in lamprey Ringer. Confocal Z-stacks were collected using a Zeiss LSM 510 laser scanning confocal attached to a Zeiss Axioskop 2FS upright microscope (10X, 0.3 NA Zeiss Plan-NEOFLUAR objective). Maximum intensity projections of the spinal cords, ranging from 2 mm proximal to 5 mm distal to the lesion center, were generated using the Zeiss LSM software. After stitching the projections together in Adobe Photoshop, the number of labeled, regenerated axons was counted at 1 mm intervals starting from the center of the lesion using ImageJ/FIJI. Resulting data were then analyzed using ANOVA statistics in Prism 8.0.0.

Knock-down of endogenous proteins in Xenopus was achieved by embryonic injection of antisense morpholino oligonucleotides (MO; Gene Tools), which bind to the target mRNA sequence that block its protein translation. The following MO sequences were used in this study: Xenopus Grp94 MO: 5′-GACCGATTGCCCAAAACTTCCTCAT-3′, Xenopus HSP90β MO: 5′-CATTGTGGGCAACTTCTGGCATC-3′, and Control MO: 5′-CCTCT TACCT CAGTT ACAAT TTATA-3′. To visualize the presence of MO in the microinjected embryos, MOs were co-injected with Alexa Fluor 488-conjugated dextran (Thermo Fisher Scientific, catalog #D22910) as a cell lineage tracer. The effectiveness of MO-mediated knock-down of endogenous proteins was validated by Western blot analyses.

The technical setup of the 2-photon microscopy was as described before [28 (link)]. The pulsed laser was tuned to 880 nm and routed through a 25× water immersion objective (N.A. 0.95, Leica). Typically, a field of 360 × 360 μm was scanned, and 40–80 μm z-stacks were acquired using a 3–6 μm z-step. The acquisition rate was set to 25.219 s intervals, with images line-averaged twice. The fluorescence signals were detected using non-descanned photomultiplier tube (PMT) detectors (Hamamatsu) equipped with 525/50 nm (for detection of Alexa Fluor 488) and 630/69 nm (for detection of dsRedII) band-pass filters (Semrock). Mice were anesthetized and imaging in the spinal cord was performed as described previously [28 (link)]. For labeling of perivascular meningeal APC, we performed local instillation of Alexa Fluor 488–conjugated dextran (10 ng/μl, 10 kDa; Life Technologies) 20 min prior to imaging, as described before [29 (link)]. Image analysis was performed as described previously [29 (link)].

Amounts of released drug were measured using a plate reader (Victor 3 Multilabel Reader, Perkin-Elmer, Waltham, MA, USA). The fluorescence intensity measured relative to the standard curve (Supplementary Figure S1) was used to calculate the amounts of doxorubicin^{15 (link)} (Sigma-Aldrich, St Louis, MO, USA) and Alexa Fluor 488 conjugated Dextran (3000 MW, anionic; Life Technologies, Carlsbad, CA, USA). Amounts of the released parathyroid hormone (1–34) were measured by absorbance (optical density) through an enzyme immunoassay kit (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). Applying a drop (20 μl) of surfactant (Triton X-100, Sigma-Aldrich) destroyed the structure of the lipid membrane, allowing the complete release of the remaining drugs into surroundings.^{16 (link)} The measured amounts of the remaining drugs in the lipid membrane were insignificant, typically within ~ 0.1 μg.

Microinjection of morpholinos was carried out as described in Rentzsch et al. (2008 (link)). Fertilised eggs were injected with 250 μM-500 μM morpholino (Gene Tools) and 40 μg/ml Alexa Fluor488-conjugated Dextran (Invitrogen) in TAE buffer. Control injections were carried out using a generic control morpholino. For morpholino sequences and control experiments for NvAth-like MO, see supplementary material Table S2. The numbers of NvElav1::mOrange⁺ cells in NvAth-like morphants and controls (Fig. 1I,J) were manually counted in maximum projections from the surface to the centre of planulae. Counting and statistical assessment was carried out using the same methods as for DAPT experiments.