Oregon green 488 bapta 5n

Ca²⁺ imaging was performed during whole cell recordings in retinal slices by filling photoreceptors with the Ca²⁺ sensitive dye Oregon Green 488 BAPTA-5N (OGB-5N, k_d = 20 μmol/L; Invitrogen) included in the patch pipette solution (500 μmol/L). OGB-5N at this concentration contributes a Ca²⁺ binding ratio (κ’) of ˜20. When included with a pipette solution containing 0.05 mmol/L EGTA, this produces a total Ca²⁺κ’ of ˜50, which is within the range of measurements we previously reported for both rods and cones (Van Hook and Thoreson 2014 (link)). Images were acquired on a Nikon E600FN microscope equipped with a spinning disk laser confocal scan head (PerkinElmer Ultraview LCI) and a cooled CCD camera (Orca ER) with UltraView Imaging Suite software (PerkinElmer). OGB-5N was excited with a 488-nm laser and emitted light passed through a 525-nm long-pass filter. The frame was cropped and binned 2 × 2 to the synaptic terminals to give quick acquisition speeds of 35 msec exposures at 35 msec intervals. Ca²⁺-dependent fluorescent signals were analyzed as ΔF/F_o (measured from a 1–2 sec prestimulus baseline) in response to depolarizing steps in voltage clamp (–79 to –19 mV) of varying duration (20–500 msec).

Antibodies used in this work are commercially available, as follows: from Abcam (Cambridge, UK), anti-H. pylori (ab20459) [24 (link)], rabbit anti-Lamp-1 (ab25030-100), mouse anti-EEA1 (ab2900), rabbit anti-Rab7 (ab50533), rabbit anti-Na⁺K⁺ ATPase (ab76020), rabbit anti-cathepsin D (ab75852), and rabbit anti-histone H3 (ab1791); from Sigma-Aldrich (Missouri, USA), rabbit anti-LC3B (L7543). Additionally, the following reagents were purchased: LysoTracker^® Red DND-99 (Invitrogen, California, USA; L7528), cholera toxin B staining kit (Invitrogen; V34404), Magic Red™ cathepsin B staining kit (Bio-Rad, California, USA; ICT937), Oregon Green™ 488 BAPTA-5N (Invitrogen California, USA; O6812), Dextran Alexa Fluor™ 568 (Invitrogen California, USA; D22912), LysoSensor™ Yellow/Blue DND-160 (Invitrogen California, USA; L7545), 3-methyl adenine (Sigma; M9281), chloroquine (Sigma; C6628), rapamycin (Sigma; R8781), bafilomycin-A1 (Sigma; B1793), and concanamycin A (Sigma; C9705).