Rabbit anti atf3

Mice were transcardially perfused with phosphate buffered saline followed by 4% paraformaldehyde (ThermoFisher). Once dissected, tissue was postfixed overnight in 4% paraformaldehyde, then overnight again in 20% sucrose (ThermoFisher) in 0.1 M phosphate buffer. The tissue was then frozen in Cryomatrix (ThermoFisher) and sectioned at 20 μm (DRG and sciatic nerve) or 50–100 μm (spinal cord). In some cases (Fig. 1), whole sympathetic or sensory ganglia were stained and imaged. All sections were blocked in 10% normal donkey serum with 0.2% Triton X-100 plus 0.02% sodium azide in PBS. Sections were incubated overnight with primary antibodies: rabbit anti-ATF3 (1:400, Santa Cruz SC-188), rabbit anti-ATF3 (1:500, Novus NBP 1-85816), rabbit anti-SCG10 (1:1000, Novus NBP 1-49461), rabbit anti-PTEN (1:400, Cell Signaling 9188), and mouse anti-PTEN (1:200, Cell Signaling 14642). Sections were incubated for 2 h with the appropriate secondary antibodies at a concentration of 1:1000: Alexa Fluor 488 donkey anti-rabbit (Invitrogen A21206), and Alexa Fluor 488 donkey anti-mouse (Jackson ImmunoResearch 715-545-151). Slides were cover-slipped with ProLong Gold with DAPI (Invitrogen).

SD rat lumbar dorsal root ganglia were subjected to the treatment described for the sciatic nerves, sectioned, and stained using the Azan-Mallory method as previously reported19 (link). The ATF-3 expression in L4-L5 DRGs was evidenced by the use of an anti-ATF-3 primary antisera (rabbit anti-ATF-3, 1:500; Santa Cruz Biotechnology), evidenced with diaminobenzidine as reported19 (link). For details, see the supplementary material (Supplementary Methods).

For immunohistochemistry, mice were transcardially perfused with 2% paraformaldehyde (PFA) in PB containing 0.2% picric acid at the appropriate endpoints. Eyes were removed from connective tissues, post-fixed in 2% PFA in PB overnight at 4 °C, and then transferred to 30% sucrose solution. For immunostaining using an anti-DINE antibody, mice were decapitated and the eyes were freshly frozen in dry ice. Both perfused and fresh frozen sections were embedded in OCT compound, cut into serial 16-μm-thick sections on a cryostat, and thaw-mounted onto silane-coated glass slides. Fresh frozen sections were fixed in 4% PFA for 10 min before immunohistochemical procedures. Both sections were washed in 0.01 M PBS, blocked with 1% BSA/0.3% triton X-100 for 30 min, washed in 0.01 M PBS, and incubated with the primary antibodies against goat anti-DINE (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-ATF3 (Santa Cruz Biotechnology), rabbit anti-GAP43 (Millipore, Billerica, MA, USA) and rabbit anti-GFP (MBL, Nagoya, Japan) antibodies at a 1:1000 dilution. After rinsing in 0.01 M PBS, sections were incubated with Alexa Flour 488 or 594-conjugated secondary antibodies (1:500, Invitrogen) at room temperature for 2 h. Following another wash, the sections were mounted and visualized by a fluorescent microscope using a × 40 objective (BZ9000, Keyence, Osaka, Japan).