Double immunofluorescence staining for interleukin (IL)-6 and activating transcription factor (ATF)-3 or glutamine synthase (GS). Representative sections through ipsilateral lumbar DRG illustrating double immunostaining for (A,D) IL-6 and (B) ATF-3, indicating neuronal bodies with injured axons, and/or (E) GS as a marker of satellite glial cells (SGC). (C) Merged picture shows that increased IL-6 immunostaining was induced by chronic constriction injury (CCI) not only in ATF-3 positive neurons (thin arrows) but also in ATF-3 free neurons (thick arrows). (F) Merged picture shows an increase in IL-6 immunofluorescence in SGC (arrowheads) surrounding large neuronal bodies. Sections were cut through ipsilateral lumbar DRG of rats subjected to unilateral CCI of the sciatic nerve for 3 days. After incubation with mouse monoclonal anti–IL-6 and rabbit polyclonal anti-ATF-3, the section was covered with affinity-purified secondary antibodies: (A) tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey anti-mouse; (B) fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit. To identify IL-6 upregulation in SGC, the section was covered with rabbit polyclonal anti–IL-6 antibody and mouse monoclonal anti-GS. Affinity-purified secondary antibodies were applied: (D) TRITC-conjugated donkey anti-rabbit; (E) FITC-conjugated donkey anti-mouse. Positions of the cell nuclei were detected by staining with Hoechst 33342. Scale bars: (A–C) 60 μm; (D–F) 40 μm.