mouse monoclonal anti gfap antibody  (Cell Signaling Technology Inc)

Journal: The Journal of Headache and Pain

Article Title: Effects of FGF4/HIF-1α axis-mediated neuronal glycolysis on neuropathic pain

doi: 10.1186/s10194-026-02362-7

Figure Lengend Snippet: FGF4 was suggested as a potential core regulator of neuronal glycolysis in CCI rats. ( A ) Heat map of differentially expressed genes. n = 4. ( B ) Ranking the top 20 differentially expressed genes in ascending order of q-value. ( C ) Construct a protein-protein interaction network of differentially expressed genes through STRING online analysis database. ( D ) Based on the global PPI network ( C ), topological analysis using Cytoscape identified FGF4 as a hub gene. For clarity, the predicted interaction lines between FGF4 and key glycolytic genes (HIF-1α, HK2, LDHA, PGK1), as well as between HIF-1α and key glycolytic genes (HK2, LDHA, PGK1), were highlighted in red. ( E ), ( F ) Protein expression levels of FGF4 in sciatic nerve and quantification data of the expression of FGF4/GAPDH in each group. n = 6. Data were analyzed using Student’s t-test. ( G ) qRT-PCR showing the expression of FGF4 mRNA in sciatic nerve. n = 6. Data were analyzed using Student’s t-test. ( H ) Immunohistochemistry of FGF4 (green)/NeuN (red), FGF4 (green)/GFAP (red) and FGF4 (green)/Iba1 (red) in the spinal dorsal horn. Scale bars, 20 μm. ( I ), ( L ) Protein expression levels of FGF4 in dorsal horn of spinal cord and quantification data of the expression of FGF4/GAPDH in each group. n = 6. Data were analyzed using Student’s t-test. ( J ) Immunohistochemistry of FGF4 (green) and NeuN (red) in the spinal dorsal horn. Scale bars, 200 μm, 50 μm. ( K ) Quantitative analysis of mean fluorescence intensity of FGF4 in neurons. n = 4. Data were analyzed using Student’s t-test. Columns represent the mean ± SD. $$ p < 0.01 vs. the S group

Article Snippet: Mouse anti-Iba1 and mouse anti-GFAP antibodies were purchased from Servicebio (China).

Techniques: Construct, Expressing, Quantitative RT-PCR, Immunohistochemistry, Fluorescence

Journal: The Journal of Headache and Pain

Article Title: Effects of FGF4/HIF-1α axis-mediated neuronal glycolysis on neuropathic pain

doi: 10.1186/s10194-026-02362-7

Figure Lengend Snippet: Intrathecal injection of FGF4-AAV increased the pain threshold of CCI rats and inhibited the expression of HIF-1α in neurons. ( A ) Schematics of AAV constructs overexpressing FGF4 or control. EGFP, enhanced green fluorescence protein; CMV, cytomegalovirus promoter; ITR, inverted terminal repeats. ( B ) Schematic diagram of experimental procedure. ( C ) Schematic diagram of AAV intrathecal injection. ( D ) Colocalization of EGFP (green) and NeuN (red), EGFP (green) and GFAP (red), and EGFP (green) and Iba1 (red) in L4-6 spinal cord segments. scale bar, 200 μm, 20 μm. ( E ) FGF4 + NeuN + , FGF4 + GFAP + and FGF4 + Iba1 + percentage statistics. ( F ) Thermal withdrawal latency (TWL). n = 15. Data were analyzed by repeated measures two-way ANOVA (Group × Day) with Bonferroni’s post-hoc tests. ( G ) Mechanical withdrawal threshold (MWT). n = 15. Data were analyzed by repeated measures two-way ANOVA (Group × Day) with Bonferroni’s post-hoc tests. ( H ), ( I ) Immunohistochemistry of FGF4 (green)/NeuN (red) and HIF-1α (green)/NeuN (red). Scale bars, 200 μm, 50 μm. ( J ), ( K ) Quantitative analysis of mean fluorescence intensity of FGF4 and HIF-1α in neurons. n = 4. Data were analyzed using one-way ANOVA followed by Tukey’s or Dunnett’s post-hoc tests. Columns represent the mean ± SD. ## p < 0.01 vs. the M + NC-AAV group

Article Snippet: Mouse anti-Iba1 and mouse anti-GFAP antibodies were purchased from Servicebio (China).

Techniques: Injection, Expressing, Construct, Control, Fluorescence, Immunohistochemistry

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: The KIBRA and p -PKMζ are activated in contralateral ACC neurons after CPTP. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, and PKMζ levels in contralateral ACC at different time points in Thoracotomy pain (A-C) or Thoracotomy no pain (D-F), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs Sham group; the data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼7 rats in each group. (G-H) The photographs of the double immunofluorescence staining show that KIBRA and p -PKMζ are only colocalized with neuron markers (NeuN) but not with astrocyte marker (GFAP) and microglia marker (Iba1) in the Sham and CPTP groups. The white arrowheads indicate the co-immunostaining. Scale bar, 100 μm. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; KIBRA, kidney and brain-expressed protein; NeuN, neuron-specific nuclear protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ.

Article Snippet: Then the samples were dehydrated in 30% sucrose, and cut into slices at 35 μm for immunofluorescence with antibodies against KIBRA (bs-11570R; Bioss), GluR1 (ab174785; Abcam), p -PKMζ (sc174785; Santa Cruz), TNF-α (BS-1857; Bioworld), IL-1β (AF-401-NA; R&D Systems, Minneapolis, MN), neuron-specific nuclear protein (NeuN, MAB377; Millipore, Burlington, VT), glial fibrillary acidic protein (GFAP, 3670s; Cell Signaling Technology, Danvers, MA), ionized calcium-binding adapter molecule 1 (Iba1, ab5076; Abcam), after incubation at 4°C overnight.

Techniques: Double Immunofluorescence Staining, Marker, Immunostaining, Binding Assay

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: GluR1 and neuroinflammation in ACC neurons are involved in CPTP. (A-F) Representative blots and columnar statistical charts show GluR1, TNF-α, and IL-1β levels in contralateral ACC at different time points in Thoracotomy pain (A-C) or Thoracotomy no pain group (D-F), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs Sham group, the data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 5∼7 rats in each group. (G-I) The photographs of the double immunofluorescence staining show that GluR1, TNF-α, and IL-1β are only colocalized with neuron markers (NeuN) but not with astrocyte marker (GFAP) and microglia marker (Iba1) in the Sham and CPTP groups. The white arrowheads indicate the co-immunostaining: scale bar, 100 μm. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GFAP, glial fibrillary acidic protein; GluR1, glutamate receptor 1; Iba1, ionized calcium-binding adapter molecule 1; IL-1β, interleukin-1β; NeuN, neuron-specific nuclear protein; TNF-α, tumor necrosis factor-α.

Article Snippet: Then the samples were dehydrated in 30% sucrose, and cut into slices at 35 μm for immunofluorescence with antibodies against KIBRA (bs-11570R; Bioss), GluR1 (ab174785; Abcam), p -PKMζ (sc174785; Santa Cruz), TNF-α (BS-1857; Bioworld), IL-1β (AF-401-NA; R&D Systems, Minneapolis, MN), neuron-specific nuclear protein (NeuN, MAB377; Millipore, Burlington, VT), glial fibrillary acidic protein (GFAP, 3670s; Cell Signaling Technology, Danvers, MA), ionized calcium-binding adapter molecule 1 (Iba1, ab5076; Abcam), after incubation at 4°C overnight.

Techniques: Double Immunofluorescence Staining, Marker, Immunostaining, Binding Assay