P 97 flaming brown puller

AII amacrine cells were visualized by injection of a recombinant adeno-associated virus serotype 2 (rAAV2) carrying a construct of green fluorescent protein (GFP) under control of a cytomegalovirus (CMV) promoter as previously described (Ivanova and Pan, 2009 (link)). The rAAV2 carries a Y444F capsid mutation for highly efficient vector transduction (Petrs-Silva et al., 2009 (link)). Briefly, mice aged postnatal day 30–60 (P30–60) were anesthetized by intraperitoneal injection of a mixture of 150 mg/kg ketamine and 15 mg/kg xylazine. Under a dissecting microscope, a small perforation was made in the temporal sclera region with a sharp needle. A total of 1.5 μl viral vector suspension in saline was injected into the intravitreal space through the perforation with a glass pipette (1B150F-4; WPI, Sarasota, FL, USA) pulled with a P-97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA). Viral vectors were packaged and affinity purified by Virovek (Hayward, CA, USA).

Pericytes were focally stimulated under an upright Nikon FN1 microscope by a current pulse (7 μA, 2 ms; Grass Technologies) using an electrode filled with Ames solution. Electrodes were pulled from borosilicate glass (WPI, 1B150F-4) with a P-97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA) and had a measured resistance of 3–5 MΩ. For consistency across all experiments, the electrode was placed near the cell body of the targeted pericyte. During focal “puff” stimulation, the electrode solution was supplemented with a vasoactive compound and delivered with picospritzer (Parker Hannifin) via a broken patch pipette positioned above the targeted pericyte. For the light stimulation experiments, the microscope’s illuminator was used to deliver a spot of light that was centered on the targeted pericyte cell body and focused on the photoreceptor cell layer. The tissue was adapted at 30 cd/m², and the stimulus was 270 cd/m². Light spot flicker (40 µm diameter, 10 Hz) was controlled by a shutter (Uniblitz, Vincent Associates). Responses to stimuli were captured on video or time-lapse photos with a microscope-mounted Sony A7s full-frame camera. Images of blood vessels were analyzed in ImageJ, using a region of interest (ROI) tracing tool. At each experimental condition, the capillary lumen cross-sections were mapped at 2 μm steps along the capillary.

Immediately prior to the experiment, cells were dissociated and an aliquot of cell suspension was transferred to a custom-made recording chamber mounted on the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). There were immersed at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, the composition of which is described above. The patch electrodes were fabricated from borosilicate glass capillaries (No. 34500; Kimble Products, Vineland, NJ, USA) on a Narishige PP-83 puller (Narishige, Tokyo, Japan) or a P-97 Flaming/Brown puller (Sutter, Novato, CA, USA), and electrode tips were fire-polished with MF-83 microforge (Narishige). Their resistances in standard pipette and bathing solutions ranged from 3 to 5 MΩ. Recordings of membrane potential or ion currents were measured in the whole-cell or cell-attached mode of the patch-clamp technique with an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) [42 (link)]. The liquid junction potentials were corrected shortly before seal formation was established.

Whole cell (current and voltage) patch-clamp recordings were performed using recording pipettes made from thick-wall borosilicate glass with filament (inner diameter: 0.75 mm; Sutter Instruments) pulled on a P-97 Flaming-Brown puller (Sutter). For current-clamp, the internal solution contained the following (in mm): 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl₂, 2 Mg2ATP, 0.2 Na3GTP, 0.1 BAPTA, and 0.02% Lucifer yellow, pH 7.3 adjusted with KOH and for voltage-clamp contained the following (in mm): 120 CsMeSO₄, 10 QX-314, 10 HEPES, 1 MgCl₂, 2.5 Mg₂ATP, 0.2 Na₃GTP, 0.1 BAPTA, 10 phosphocreatine, pH 7.3 adjusted with CsOH. Osmolarity for both solutions were in the range 287–295 mOsm. Recordings were discontinued if access resistance was >20 MΩ at the beginning of whole-cell recording with typical access resistances of 10-20 MΩ. Membrane capacitance (C_m) for SACs was 6–10 pF and for PGCs were 5–8 pF. All data were acquired with pCLAMP 9 software using a MultiClamp 700A amplifier, digitized with a Digidata 1322A A/D board (Molecular Devices), low-pass filtered online at 2 kHz (voltage-clamp, sampling rate of 5 kHz) or 10 kHz (current-clamp, sampling rate of 40 kHz).

Emitters
for nESI experiments were produced in-house from borosilicate thin-wall
glass capillaries with a starting outer diameter (OD) of 1 mm and
an inner diameter (ID) of 0.78 mm (Harvard Apparatus, Holliston, MA).
Borosilicate capillary emitters were pulled to ∼10 μm
ID for lipid experiments and ∼1–2 μm ID for protein
experiments using a Sutter Instruments P-97 Flaming Brown Puller (Novato,
CA). Parameters for the puller programs are provided in Table S2. Samples were loaded into the nESI emitter
(2–8 μL) using a gel loader pipet, and the emitters were
mounted on an x, y, z manual linear stage (Thorlabs, Newton, NJ) to
control their position relative to the inlet sampling cone. The emitter
tip was held between 7–10 mm away from the inlet orifice. Ions
were generated in positive ion mode in all cases, although negative
ion mode is also possible.