Observer z1 imaging system

A 3-mm diameter skin biopsy was taken under subcutaneous anaesthesia on the ventroradial side of the proximal phalanx of the index finger innervated by the median nerve. The biopsy was fixed in fresh periodate-lysine-paraformaldehyde for 30 minutes. The tissue was then washed in phosphate buffer and stored for 2 to 3 days in sucrose in phosphate buffer. After embedding in optimal cutting temperature gel, the tissue was frozen and stored at −80°C. Staining was performed using a previously described free-floating method,^{46 (link)} using protein gene product 9.5 (PGP 9.5 Ultraclone, Isle of Wight, United Kingdom, 1:1000; Zytomed, Berlin, Germany 1:200) and myelin basic protein (Abcam, Cambridge, United Kingdom 1:500) as primary antibodies and Cy3 (Stratech, Ely, United Kingdom 1:1000) and Alexa Fluor 488 (Abcam, 1: 500) as secondary antibodies.
Intraepidermal nerve fibre density (IENFD) was quantified in 50-μm skin sections using an Axio LSM 700 microscope with an Observer Z1 imaging system (Zeiss, Cambridge, United Kingdom) by determining the amount of fibres per millimeter epidermis according to current guidelines.^{30 (link)} We also quantified dermal innervation by evaluating the number of Meissner corpuscles per millimeter epidermis, the percentage of PGP⁺ dermal nerve bundles containing MBP, and the mean nodal length as previously reported.^{46 (link)}

Hind footpa skin biopsies were collected, immersed for 6–8 h at 4 °C in Zamboni’s fixative (2% paraformaldehyde, 0.2% picric acid in 0.1 M phosphate buffer), rinsed in 30% sucrose in PBS solution overnight, cryoembedded in mounting media (OCT), and sectioned at 14 μm thick before being processed for immunohistochemistry. Sections were incubated at 4 °C for 16–24 h with rabbit anti-primary PGP9.5 antibody (1:200; Sigma-Aldrich, Singapore). Sections were then rinsed 3 times in PBS and incubated with secondary anti-rabbit AlexaFluor 488 antibody, before being rinsed and mounted. To confirm that there was no nonspecific immunoreaction, additional sections were incubated with primary or secondary antibodies alone. Fluorescent images were collected on an Olympus microscope with an ObserverZ1 imaging system (Zeiss, Oberkochen, Germany). Six sections were measured for each footpad, and the average linear density of IENF was calculated according to current guidelines [107 (link)].

Neurite outgrowth was assessed as previously described.¹⁴ (link) Briefly, mature iPSCdSNs were enzymatically treated for 30 min with 0.1% Trypsin (ThermoFisher), followed by mechanical dissociation with a fire polished glass pipette. Single cells were re-plated onto matrigel treated coverslips at low density in the presence or absence of neurotrophic factors (NGF, GDNF, BDNF, NT3, all at 25ng/ml). 24 hours after replating, iPSCdSNs were fixed in 1% PFA and immunostained with NF200. After immunostaining, the coverslips were mounted with VectorShield on slides and sealed. To determine neurite outgrowth, 20x magnified images of all iPSCdSNs on the coverslip were taken on an Observer Z1 imaging system (Zeiss) and analyzed for neurite length using WIS-Neuromath software.⁶⁴ (link) Total neurite length, longest branch and branch points were automatically determined using the same selection parameters for all experiments. The resultant values were averaged across differentiation per iPSC line (3 control, 3 HSN1).