Hyrax c60 cryostat

Thymus tissue samples were cryosectioned for immunohistochemistry using a Hyrax C60 cryostat (Zeiss). Thymus sections (10 μm) were fixed with 2% (wt/vol) paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4, and acetone, washed in PBS, and blocked with PBS supplemented with 0.1% Triton X-100 and 4% normal goat serum. Subsequently, sections were incubated with the following primary antibodies (diluted in blocking solution) overnight at 4 °C: rat anti-GM-CSF antibody (BD Pharmingen, clone BVD2-21C11, 1:50), mouse anti-CD4 antibody (Biolegend, clone RPA-T4, 1:50) and rabbit anti-CD3 (NOVUS, clone SP7, 1:100). Sections were then washed in PBS and incubated with AF647-labeled goat anti-rat, AF488-labeled goat anti-mouse and AF555-labeled goat anti-rabbit secondary antibodies (Life Technologies, 1:500) overnight at 4 °C. Sections were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Fluorescence photomicrographs were captured with a SP5 Leica confocal laser scanning microscope (SP5; Leica) equipped with argon and helium lasers using the 40 × objective lens (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane).

Mice were transcardially perfused with 4% ice-cold paraformaldehyde (PFA, in 0.1 M sodium phosphate buffer, pH 7.4). Lumbar spinal cord and brain were immediately dissected and postfixed for 2.5 h with 4% PFA on ice. Postfixed tissue was briefly washed with 0.1 M sodium phosphate buffer (pH 7.4) and then incubated in 30% sucrose (in phosphate buffered saline – PBS) overnight at 4°C for cryoprotection. Cryoprotected tissue was cut at 25 μm or 40 μm (spinal cord or brain, respectively) on a Hyrax C60 Cryostat (Zeiss), mounted on superfrost plus glass slides and then incubated with the respective combinations of primary antibodies in 1% donkey serum in PBS over-night at 4°C. After brief washes in PBS, sections were incubated with the respective secondary antibodies for 2 h at room temperature and briefly rinsed in PBS, before mounting with coverslips and DAKO fluorescent mounting media (Dako). Secondary antibodies raised in donkey were purchased from Jackson Immuno Research. All primary antibodies used are listed in Table 1.

Mice were transcardially perfused with 4% ice-cold paraformaldehyde (in 0.1M sodium phosphate buffer, pH 7.4). Lumbar spinal cords, brains and dorsal root ganglia (DRGs) were immediately dissected and post-fixed for 2.5 h with 4% paraformaldehyde (PFA) on ice. Post-fixed tissue was briefly washed with 0.1M sodium phosphate buffer (pH 7.4) and then incubated in 30% sucrose (in PBS) overnight at 4°C for cryoprotection. Cryoprotected tissue was cut at 25 μm, 40 μm or 16 μm (spinal cord, brain, or DRGs respectively) on a Hyrax C60 Cryostat (Zeiss, Oberkochen, Germany), mounted on superfrost plus glass microscope slides and then incubated with the respective combinations of primary antibodies in 1% donkey serum in phosphate buffered saline (PBS) overnight at 4°C. After brief washes in PBS, sections were incubated with the respective secondary antibodies for 2 h at room temperature and briefly rinsed in PBS, before mounting with coverslips and DAKO fluorescent mounting media (Dako, Carpinteria, CA, USA). Secondary antibodies raised in donkey were purchased from Jackson Immuno Research (West Grove, PA, USA). All primary antibodies used are listed in the KRT.