Ab115513

Rat sciatic nerves were fixed in situ in 4% PFA for 10 min, dissected, embedded in O.C.T. Compound (Tissue Freezing Medium; Solarbio, Shanghai, China). Sciatic nerve cryosections (5-μm thick) were incubated with acetone for 10 min at − 20 °C, washed in PBS/0.1% Tween 20, blocked for 30 min at room temperature (RT) in blocking buffer (0.3% Triton X-100/10% goat serum/phosphate buffer saline ¼ PBS), and incubated with primary antibodies overnight at 4 °C in blocking buffer. Sections were then washed 3 times in blocking buffer, and sections were incubated with secondary antibodies for 1 h at RT in the dark. Sections were washed again, incubated with DAPI for 5 min at RT, washed and mounted in Citifluor (Agar Scientific).
The primary antibodies used for IF were as follows: neurofilament (1:1000, Abcam, ab8135), SCG10 (1:500, Abcam, ab115513), IBA1 (1:100, Abcam, ab178847), and CD68 (1:100, Abcam, ab125212). All secondary antibodies were also purchased from Abcam. Images were acquired using a Leica TCS SP-II confocal microscope.

Diabetes-mimicking αTC1-6 cells were generated as mentioned above and cultured on coverslips. Experiments were done in the presence or absence of lysosomal inhibitor, BFA1, in serum-free DMEM containing 16.7 mM glucose and 0.1% BSA for 2 h. Cells were then washed 3X in PBS, fixed in 2% paraformaldehyde for 30 min, washed 5X in PBS, and incubated with blocking buffer (2% BSA in PBS containing 0.05% Tween 20) for 1 h. Cells were then incubated with appropriate primary antibodies prepared in blocking buffer against glucagon (mouse anti-glucagon antibody, Cat # ab10988, Abcam; 1:1000 or rabbit anti-glucagon antibody, Cat# ab92517, Abcam; 1:25), Stathmin-2 (goat anti-SCG10 antibody, Cat # ab115513, Abcam; 1:250), or Lamp1 (mouse anti- Lamp1 antibody, Cat # ab25630, Abcam; 1:50). Following an overnight incubation at 4°C, coverslips were washed with PBS, and incubated for 2 h in the dark at RT with appropriate secondary antibodies (donkey anti-mouse IgG Alexa Fluor 488, Cat# A-21202, Molecular Probes; donkey anti goat IgG Alexa Fluor 555, Cat# A-21432, Molecular Probes; donkey anti-rabbit IgG Alexa Fluor 647, Cat# A-31573, Molecular Probes). Then, coverslips were washed with PBS, stained with DAPI, and mounted on glass slides using ProLong antifade mountant. Image acquisition and analysis was done as described above. Experiments were repeated four times using freshly thawed cells.