Dylight549 conjugated streptavidin

For dye injections (Fig. 1), retinas were isolated from the eyecup, bisected and embedded in 2% agar-agar in Ringer's solution. Vertical sections (200 µm) were prepared as described previously (Dedek et al., 2006 (link)). AII amacrine cells were injected, under visual control, with microelectrodes (120–220 MΩ) filled with 7.5 mM Alexa-Fluor-594 potassium hydrazide (Invitrogen, Karlsruhe, Germany) diluted in 0.2 M KCl, pH 7.4. Cells were filled for 3–6 min. Tracer injections and whole-mount dye injections (Figs 4–6) were performed as described previously (Dedek et al., 2006 (link); Shelley et al., 2006 (link)) using light-adapted mice. Retinal quarters were mounted, ganglion-cell side up, on black filter paper and incubated for 20 min in 0.1 mM DAPI diluted in Ringer's solution to visualize the nuclei of AII cells. Microelectrodes (120–180 MΩ resistance) were filled with 5 mM Alexa Fluor 488 and 4% (w/v) neurobiotin (Vector Laboratories, Burlingame, CA) or 7.5 mM Alexa Fluor 555 or 594. The Alexa dye was iontophoresed with 0.5 nA square pulses of 750 ms at 1 Hz for 1–2 min to visualize morphology; the current was reversed to inject positively charged neurobiotin (6–7 min), which was visualized with DyLight549-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA).

In vivo cysteine S-Nitrosylation levels were measured using biotin derivatization as previously described^{45 (link)}. Briefly, 20 µm parasagittal brain sections (every 20th) of 12-week-old C57BL/6, Ncf1^–/–, or Igfbp2^–/– mice were fixed in 4% paraformaldehyde in PBS and washed three times with PBS containing 0.4 mM EDTA and 40 µM neocuproine. Free thiol groups were then blocked with 40 mM N-ethylmaleimide (NEM) in PBS containing 0.4 mM EDTA, 0.04 mM neocuproine, and 2.5% SDS for 30 min. Sections were washed three times and then incubated with 1 mM sodium ascorbate in PBS for 15 min to reduce S-nitrosylated proteins. Newly reduced cysteine residues were then labeled with 0.1 mM N-(3-Maleimidopropionyl)biocytin (MPB) in PBS for 30 min. After washing, sections were incubated with DyLight 549-conjugated streptavidin (1:250, Jackson Immunoresearch Labs) for 30 min. Nuclei were counterstained with Hoechst 33342 and coverslips were mounted in Aquamount. Slides from different groups were stained and imaged, in parallel. Quantification was performed by Metamorph software at the same threshold for all images.