Fig 1: (A) Double immunofluorescence revealed that GFAP was co-localized with α2AR and TLR4 immunoreactivity in the spinal dorsal horn of the CFA group at 3 days. Arrowheads indicate double-labeled cells. (B) Co-immunoprecipitation assay. α2AR was detected by with specific antibodies to TLR4, and TLR4 was also detected with specific antibodies to α2AR, as was the positive control. However, when IgG was used as a negative control, no immunoreactivity was detected. Magnification, ×20. IgG, immunoglobulin G; GFAP, glial fibrillary acid protein; TLR, Toll-like receptor; α2AR, α 2-adrenoceptors.
Fig 2: Fluorescence immunostaining for cellular localization of CX3CR1 in the injured spinal cord after SCI. (A) Co-immunostaining of CX3CR1 (green) and the neuronal marker NeuN (red). Note that CX3CR1 was not localized in the NeuN positive cells (neurons). (B) Co-immunostaining of CX3CR1 (green) and the microglial marker CD11-B (red) after SCI. The arrows indicate the co-localization (yellow) of CX3CR1 and CD11-B. (C) The corresponding quantitative analysis is shown. (D) Co-immunostaining of CX3CR1 (green) and the astrocyte marker GFAP (red) in injured spinal cord tissues. The arrows indicate the co-localization (yellow) of CX3CR1 and GFAP. The corresponding quantitative analysis is shown in (E). The data was represented as the mean ± SD. **P<0.01 and ***P<0.001 vs. sham. Scale bar=50 μm. n=5. Me, methylprednisolone; ns, not significant; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury; CX3CL1, C-X3-C motif chemokine ligand 1CX3CR1, C-X3-C motif chemokine receptor 1; DAPI, 4′,6-diamidino-2-phenylindole; CD, cluster of differentiation.
Fig 3: Expression of GFAP and the development of mechanical and thermal hypersensitivity in CFA-induced MA. Effects of i.t. injection with DEX (2.5 µg) on (A) paw withdrawal mechanical threshold and (B) paw withdrawal thermal latency of the ipsilateral hind paws prior to the behavioral test on day 1–3 after surgery. **P<0.05, SHAM vs. CFA; #P<0.01, ##P<0.05, CFA vs. DEX; £P<0.01, ££P<0.05, CFA vs. EGCG; &&P<0.05, B vs. DEX; $$P<0.05, B vs. EGCG. (C and D) Western blot analysis of GFAP in the spinal dorsal horn of rats in the Sham, CFA, DEX, EGCG and B groups. Effects of intrathecal injection with DEX, BRL44408 and EGCG over 3 consecutive days on the GFAP levels in the spinal dorsal horn induced by MA on (C) day 3 and (D) day 7. GAPDH served as loading control. **P<0.05, SHAM vs. CFA; #P<0.01, DEX vs. CFA; £P<0.01, DEX vs. B; &&P<0.05, EGCG vs. CFA; $P<0.01, $$P<0.05, EGCG vs. B. Groups: Sham, i.a. injection with 50 µl sterile saline and i.t. injection with 20 µl sterile saline for 3 consecutive days; CFA, i.a. injection with 50 µl CFA; DEX, i.a. injection with 50 µl CFA and i.t. injection with DEX (2.5 µg/20 µl) for 3 consecutive days; B, i.a. injection with 50 µl CFA and pre-treatment with BRL44408 (15 µg/20 µl) 30 min prior to i.t. injection with DEX 2.5 (µg/20 µl) for 3 consecutive days; EGCG, i.a. injection with 50 µl CFA and i.t. injection with EGCG 30 µg for 3 consecutive days. CFA, complete Freund's adjuvant; MA, monoarthritis; DEX, dexmedetomidine; i.t., intrathecal; i.a., intra-articular; EGCG, epigallocatechin gallate; B, BRL44408; GFAP, glial fibrillary acid protein.
Supplier Page from MilliporeSigma for Anti-GFAP antibody produced in mouse