Immunofluorescence Analysis of Spinal Cord in Diabetic Mice

Immunohistochemistry assays were performed on paraffin-embedded 7-µM-thick microtome sections of lumbar (L4–L6) spinal cords that were removed from diabetic mice on day 7 after STZ administration. Section preparation and immunofluorescence staining were performed as described by Chmielarz et al. (43 (link)). Briefly, after deparaffinization and subsequent antigen retrieval (microwave method with citrate buffer), sections from STZ-treated mice were incubated for 30 min in 5% normal pig serum (Vector Labs) in PBST buffer (0.2% Triton X-100 in PBS). Sections were incubated overnight at 4°C with the following primary antibodies: anti-CCL3 (1:50, ThermoFisher Scientific), anti-CCL9 (1:50, Bioss), anti-CCR1 (1:50, Novus), anti-CCR5 (1:50, Novus), anti-NeuN (1:200, Millipore), anti-Iba1 (1:50, Abcam, UK), and anti-GFAP (1:500, Millipore). Antigen-bound primary antibodies were visualized with anti-rabbit Alexa-488, anti-mouse Alexa-594, or anti-chicken Alexa-594 (1:100; Invitrogen) secondary antibodies. Stained sections were examined and photographed under a fluorescence microscope (Nikon Eclipse 50i). The dorsal part of the lumbar spinal cord was visualized in representative images.