Lsm 710 axio examiner z1 microscope

Experiments were performed on an upright LSM 710 Axio Examiner Z1 microscope equipped with a 100×/1.30 NA Oil UV objective (Carl Zeiss, Oberkochen, Germany). Images were acquired with Zen 2009 software (Carl Zeiss).

In vivo 2-photon imaging was performed as previously described (58 (link), 59 (link)). Briefly, animals were anesthetized by 1 mg/kg medetomidine and 100 mg/kg ketamine, tracheally intubated, ventilated, and stabilized in a custom-made heated microscope stage. Body temperature was monitored with a rectal thermometer (Telemeter Electronic GmbH) and was kept constant at 36–37°C. The lower thoracic SC was accessed by performing a laminectomy at level Th12/L1 as previously described (59 (link)). The animals were imaged at the onset and peak of disease, i.e., between day 6 and day 13 after transfer. Blood vessels were visualized by i.v. injection of fluorescently labeled 2000 kDa dextran. Intravital 2-photon recordings were performed with an LSM710/Axio Examiner Z1 microscope (Carl Zeiss) and a greater than 2.5-watt Ti:Sapphire Chameleon Vision II Laser (Coherent GmbH). Stacks were acquired with 4 μm intervals apart on the z axis and scan size of 512 × 512 pixels. Each stack was acquired for 58 cycles with a 32-second time interval and 2 line averaging. Four-dimensional stacks were analyzed with Imaris software (Bitplane). Motility parameters were exported into Microsoft Excel and plotted using GraphPad Prism 8.0.