immunofluorescence  (Novus Biologicals)

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 is a key gene in maintaining microglial homeostasis during cognitive impairment in TN rats. ( a ) Volcano plot of transcriptomic differentially expressed genes (DEGs). ( b ) Protein–protein interaction network of cognition-related DEGs. ( c ) Heatmap of cognition-related DEGs. ( d ) mRNA expression of Nr4a1 in the hippocampus measured using RT-qPCR ( n = 3). ( e ) Protein expression of Nr4a1 was analyzed using western blotting ( n = 6). ( f - g ) Representative immunofluorescence images and quantitative analysis of Nr4a1 expression in the hippocampus ( n = 6). Scale bar = 20 μm. ( h ) Flowchart of the experimental timeline for in vivo Part II. ( i ) Changes in the facial mechanical pain threshold ( n = 8). ( j ) Schematic diagram of the empty vector control and Nr4a1 -overexpressing AAV constructs. ITR, inverted terminal repeat; CX3CR, ZsGreen and Nr4a1 promoter. (k) Image showing AAV- Nr4a1 virus infusion into the hippocampus. Scale bar = 200 μm. Data are expressed as mean ± SD. # P < 0.05, ^ P < 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, In Vivo, Plasmid Preparation, Control, Construct, Virus

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 overexpression alleviates microglial overactivation and its mediated neuroinflammation in the hippocampus of TN rats. ( a - d ) Representative immunofluorescence images and quantitative analysis of Iba1 expression, microglial number, and morphological changes in the hippocampal CA1 region ( n = 6). Scale bar = 20 μm. ( e - k ) Protein expression levels of Iba1, TNF-α, IL-6, IL-1β, IL-4, and IL-10 were analyzed using western blotting ( n = 6). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Over Expression, Immunofluorescence, Expressing, Western Blot

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 overexpression improved neuronal autophagy and synaptic plasticity in the hippocampus of TN rats. ( a ) Representative TEM images of hippocampal neuronal ultrastructure (orange arrows indicate autophagosomes) ( n = 3). Scale bar = 2 μm. ( b - d ) Protein expression levels of LC3-II and p62 were analyzed using western blotting ( n = 6). ( e - j ) Protein expression levels of BDNF, TrkB, SYN, and PSD95 were analyzed using western blotting ( n = 6). ( k ) Representative Golgi staining images of hippocampal dendritic spines (different arrow types indicate spine morphologies). Scale bar = 10 μm. ( l - t ) Quantitative analysis of total dendritic spine density, mature spine density, and immature spine density in the hippocampus ( n = 6). Data are expressed as mean ± SD. ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Over Expression, Expressing, Western Blot, Staining

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Co-IP/MS identified Akt2 binding to Nr4a1 . Restoring Nr4a1 expression rescued downregulated PI3K/Akt2 signaling in the hippocampus of TN rats and in an LPS-stimulated BV2 cell model. ( a ) Flowchart of the Co-IP/MS experiment. ( b ) Potential protein interactors of Nr4a1 predicted using the STRING database. ( c ) Silver staining of Co-IP products (target protein: Nr4a1 ). ( d ) Protein identification results from mass spectrometry analysis of Co-IP products. ( e ) Molecular docking models of Nr4a1 with different Akt isoforms. ( f ) Representative immunofluorescence images of Nr4a1 and p-Akt2 in each group of cells. Scale bar = 20 μm. ( g - j ) Protein expression levels of p-PI3K, PI3K, p-Akt2, Akt2, p-mTOR, and mTOR in BV2 cells from each group were analyzed using western blotting ( n = 6). ( k - n ) Protein expression levels of p-PI3K, PI3K, p-Akt2, Akt2, p-mTOR, and mTOR in the hippocampus of rats from each group were analyzed using western blotting ( n = 6). Data are expressed as mean ± SD. *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Expressing, Silver Staining, Mass Spectrometry, Immunofluorescence, Western Blot