Ax10 fluorescence microscope

Cultured DRG neurons, Schwann cells, and fibroblasts were first fixed in 4% paraformaldehyde (Sigma-Aldrich) for 30 minutes. They were then incubated with bovine serum albumin (5%; from Invitrogen) for 1 hour at room temperature to block nonspecific binding. Then, the cells were incubated with antibodies to the fibroblast marker Thy-1 cell surface antigen (anti-CD90) (mouse, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab225, RRID: AB_2203300), the Schwann cell marker S100 calcium-binding protein (Anti-S100) (rabbit, 1:1000, Sigma, Cat# SAB5500172) and the DRG neuron marker β3-tubulin (rabbit, 1:1000, Cell Signaling, Danvers, MA, USA, Cat# 5568S) at 4°C for 12–16 hours. Then, the cells were incubated with Alexa Fluor 594 goat anti-mouse IgG (1:400, Abcam, Cat# ab150116, RRID: AB_2650601) and goat anti-rabbit IgG H&L Alexa Fluor 488 (1:500, Abcam, Cat# ab150077, RRID: AB_2630356) secondary antibodies for 2 hours at room temperature. Nuclei were labeled with Hoechst 33342 (1:1000, Abcam, Cat# ab145597). The morphology of Schwann cells and fibroblasts was observed under a TCS SP2 confocal microscope and a Zeiss-ax10 fluorescence microscope (Carl Zeiss). Cell type and axon length statistics were confirmed using ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012).

The remaining paraffin section slides (n=77) were stained using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay kit (in situ cell death detection kit; Roche, Mannheim, Germany). In brief, after deparaffinization and rehydration, the sections were rinsed with DPBS and treated with 0.8% proteinase K (Dako, Glostrup, Denmark) at room temperature for 15 minutes, and then incubated with a TUNEL reaction mixture for 1 hour in a 37℃ humidified chamber in the dark. After washing, the sections were mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) and visualized under an inverted Zeiss AX10 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Green fluorescence was detected in the follicular cells containing fragmented DNA at an excitation wavelength of 450 to 500 nm and a detection wavelength of 515 to 565 nm. Blue fluorescence was visualized in the counterstained normal follicular cells at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. As shown in Figure 2, when green fluorescence was detected in more than 30% of a single follicle, the follicle was regarded as apoptotic, according to previously published guidelines [7 (link)9 (link)22 (link)]. Three slides could not be counted due to staining errors.