Stemi 2000 microscope

Vessel diameter was recorded with the help of a micrometer coupled with a STEMI 2000 microscope (Zeiss; North York, ON). Measurements were taken after every intervention when the myograph had been sealed inside of the barometric pressure-controlled chamber. A transparent lid allowed for micrometer measurements. Desiccants lined the inner walls of the chamber to ensure visibility of the vessels was not diminished due to increased humidity in the chamber.

iPSC-RPE cells were injected into the subretinal space of Mertk^–/– mice (129 genetic background) and BALB/cJ albino mice. Cultures of iPSC-RPE cells were washed thoroughly with PBS before enzymatic dissociation with TrypLE™. BSS PLUS™ (0065080050; Alcon Laboratories) was added to create a suspension at a concentration of 50,000 cells/μl. Mice (postnatal day (P) 10–16) were anesthetized by isoflurane inhalation. Their pupils were dilated with a drop of 1% (w/v) Atropine Sulfate ophthalmic solution (17478-215-02; Akorn Pharmaceuticals), and the corneas were kept moist with Hypromellose ophthalmic demulcent 2.5% solution (51394-315-15; Wilson Ophthalmic Corp.). A 1-μl suspension of iPSC-RPE cells was injected into the subretinal space of each eye, under a Zeiss Stemi 2000 microscope, as described previously [26 (link)]. Ophthalmic ointment (Neomycin & Polymyxin B sulfates and Dexamethasone, 61314-631-36; Falcon Pharmaceuticals) was applied to each eye immediately following injection. Cyclosporine (200 mg/l, 0078-0109-61; Novartis) was added to the drinking water of the dam from 1 day prior to the injection until the pups were weaned at P28. Mice were kept on a 12-h dark/12-h light cycle. For experiments concerning phagosomes, they were killed between 15 and 30 min after lights on.

All procedures were approved by the Johns Hopkins University Animal Care and Use Committee, protocol no. FI15M197. Zebrafish were raised in the FINZ center at the Institute for Genetic Medicine (Johns Hopkins University) as described previously (Westerfield 2000 ). Zebrafish were maintained at 28°. Male and female Tubingen zebrafish were placed in the same breeding tank in the morning and embryos were collected 30 min later. One hundred embryos were then injected at the 1-4 cell blastula stage using a Zeiss Stemi 2000 microscope and PV820 Pneumatic picopump injector. All genes were injected at 50pg or 100pg and most were injected a second time at a different dose (Table S2). All of those producing a phenotype were re-injected at 100pg. Embryos were raised to 5 days post fertilization and then phenotyped using a Nikon SMZ1500 microscope and imaged with NIS Elements Imaging Software. After imaging, embryos were fixed in 4% PFA overnight then transferred to 100% Methanol for storage at -20°. For low dosage experiments, SOD1 and RRP1 were injected at 2pg, 5pg and 10pg and examined at 5 dpf.

Mycobacterial strains (RvS, ΔRv0950c and ΔRv0950c::Rv0950c) were grown to mid-exponential phase (OD_600nm ~ 0.5) in 7H9 media (Middlebrook, Sigma) supplemented with OADC and 0.05% Tween80. Ten microliters of each strain was aliquoted onto solid 7H11 media (Middlebrook, Sigma) and allowed to dry completely. Plates were then incubated for 21 days at 37°C. Images of the entire colony were captured using a digital camera (Nikon) and higher magnifications of the colony center or edge were captured using a stereo microscope (Zeiss Stemi 2000 microscope running Zen 2011, blue edition software).

Five- to seven-week-old mice were anesthetized with isoflurane inhalation and placed under a Stemi2000 microscope (Carl Zeiss Microscopy, Rueil Malmaison, France). The incision area was shaved and cleaned using betadine solution. After incision, the gluteus superficialis and biceps femoris muscles were separated to reveal the cavity traversed by the sciatic nerve. The nerve was lifted out using a spatula, and a thin glass needle filled with viral solution (8 μL) was introduced into the nerve with a micromanipulator. This solution was injected over 30 min with short pressure pulses using a Picospritzer III (Parker Hannifin, Contamine-sur-Arve, France) coupled to a pulse generator. After injection, the nerve was replaced into the cavity, the muscles were readjusted, and the wound was closed using clips (for further details, see Gonzalez et al., 2014 [25 (link)]).