Lumbar spine tissues were freshly isolated and paraffin-embedded after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (5 μm). After preparation of the slides as described previously [22 (link)], tissue samples were incubated with anti-IBA1 antibody (Cell Signaling, 17198, Danvers, MA, USA) at 4 °C overnight. Samples were then incubated with goat anti-rabbit Alexa Flour 594 (Invitrogen, A32740, Carlsbad, CA, USA) for 1 h at room temperature. Tissues were mounted with Slow Fade (Invitrogen, s2828, Carlsbad, CA, USA), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with Zeiss laser scanning microscope (LSM) 710. Fluorescence intensity was determined by using Image J software, Image J Version Fiji, https://imagej.net/software/fiji/ , accessed on 10 November 2022). This method determines the corrected total fluorescence by subtracting out background signals, which is useful for comparing the fluorescence intensity between cells or regions.
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