Journal: Frontiers in Pharmacology

Article Title: Changes in the excitability of the medial parabrachial nucleus neurons during the chronic phase of pilocarpine-induced epilepsy in mice

doi: 10.3389/fphar.2025.1474254

Figure Lengend Snippet: Pilocarpine significantly increases glial fibrillary acidic protein (GFAP) expression in the chronic phase of epilepsy in mice (A) Immunofluorescence staining of GFAP in the medial parabrachial nucleus (MPB) in slices obtained from animals in the saline, pilocarpine (Pilo), and Pilo + valproic acid (VPA) groups. The GFAP protein is stained in red, and 4′,6-diamidino-2-phenylindole-stained nuclei are stained in blue (B) Western blot detection of GFAP expression in the three experimental groups [ (C) , left] Statistical analysis of GFAP expression assessed using immunofluorescence staining [ (C) , right] Statistical analysis of GFAP expression assessed using Western blot. Statistical analysis of GFAP expression was assessed using Student’s t-test and one-way ANOVA. Statistical significance of differences is indicated as follows: *** P < 0.001. Data are shown as the mean ± standard error of the mean.

Article Snippet: The slices were then incubated overnight at 4°C with the primary rabbit monoclonal antibody against FosB (1:200; #2251; Cell Signaling Technology) or the primary rabbit monoclonal antibody against GFAP (1:200; #80788; Cell Signaling Technology, Danvers, MA, United States of America).

Techniques: Expressing, Immunofluorescence, Staining, Saline, Western Blot

Journal: Genes

Article Title: Hwanhon Decoction Ameliorates Cognitive Impairment and Suppresses Neuroinflammation in a Chronic Cerebral Hypoperfusion Mouse Model: Involvement of Key Genes Identified by Network Pharmacology

doi: 10.3390/genes16070746

Figure Lengend Snippet: Observation of expression of GFAP-positive astrocytes and CD68-positive microglia using IF staining. ( A ) GFAP expression in the cerebral cortex, including the corpus callosum, significantly increased after BCAS-induced CCH, and this increase in expression was inhibited by HHex treatment. ( B ) BCAS-induced CCH or HHex treatment did not affect CD68 expression levels. Data are expressed as mean ± standard error ( n = 5). ** p < 0.01, *** p < 0.001 vs. compared group.

Article Snippet: After 1 h of reaction at 25 °C with blocking buffer (5% bovine serum albumin), the blocking buffer was cleaned, and diluted primary antibodies of cluster of differentiation 68 (CD68+), ionized calcium-binding adaptor molecule 1, neuronal nuclear protein, and glial fibrillary acidic protein (GFAP) (#97778, #17198, #94403, and #12389, respectively; Cell Signaling Technology, Danvers, MA, USA) were added and reacted for 12 h at 4 °C.

Techniques: Expressing, Staining

Journal: Bioengineering

Article Title: Engineered Dual Antioxidant Enzyme Complexes Targeting ICAM-1 on Brain Endothelium Reduce Brain Injury-Associated Neuroinflammation

doi: 10.3390/bioengineering11030200

Figure Lengend Snippet: Representative images and quantitative evaluation of post-TBI neuropathological indices for neuronal cell death, astrocyte and microglial activation in animals treated with or without dual conjugates. Mice were placed in the following groups prior to brain harvesting and immunohistochemistry: naïve (craniotomy only but without impact), moderate CCI-TBI and moderate CC-TBI + 100 ng/kg of dual conjugate administered i.v. ( A ) NeuN staining identifies viable neurons within the cerebral cortex and lack of DAB+ staining for NeuN demonstrates the substantial neuronal loss at 48 h following CCI-TBI. The combination of mechanical strain, oxidative stress and inflammatory responses inevitably results in cell death. However, a rescue of viable neurons can be observed with the presence of dual conjugate administered soon after the TBI was induced. Astrocyte activation was determined by GFAP expression. CCI-TBI shows significant increase at 48 h following CCI-TBI, which is attenuated with the presence of the dual conjugate. Similarly, evaluation of activated microglial by phenotype and augmentation of expression of IBA-1 is clearly seen in the TBI animal. Dual conjugates also reduced the degree of microgliosis. Scale bar 50 microns ( B ) Bar graphs show the imaging quantification of the neuropathological indices from the groups outlined above. Data are presented as means ± SEM. For ( B – D ), one-way ANOVA with Dunnett’s test for multiple comparisons was used. ** p < 0.001, N.S = not significant.

Article Snippet: All sections were incubated in primary antibody prepared in Dako Antibody Diluent either for 1 h at RT (NeuN, Iba1) or overnight at 4 °C (GFAP) at the following dilutions: NeuN (1:500, Abnova Corp., Walnut, CA, USA), Iba-1 (1:400, (Fujifilm Wako Chemicals Inc., Richmond, VA, USA), GFAP (1:2000, Cell Signaling Technologies Inc., Danvers MA, USA).

Techniques: Activation Assay, Immunohistochemistry, Staining, Expressing, Imaging

Journal: Cells

Article Title: Phagocytic Activities of Reactive Microglia and Astrocytes Associated with Prion Diseases Are Dysregulated in Opposite Directions

doi: 10.3390/cells10071728

Figure Lengend Snippet: Preparation of adult primary microglia cell cultures. ( A ) Co-immunostaining of PMCs for microglia (CD11b, green) and astrocytes (GFAP, red), oligodendrocytes (Olig2, red), or neurons (NeuN, red). Cell nuclei are stained with DAPI (blue). Images are representatives of three independent cultures, each from an individual control animal. Scale bars = 25 μm. ( B ) Representative Western blots (left) and densitometric analysis of Iba1 expression levels (right) in CT-PMCs and 22L-PMCs normalized per expression of β-actin. Data are expressed as mean ± SEM, n = 3 independent cultures isolated from individual animals, ** p < 0.01 (two-tailed, unpaired student t-test).

Article Snippet: The following antibodies were used: horseradish peroxidase (HRP), conjugated secondary anti-mouse IgG (cat. A9044), anti-rabbit IgG (cat. A0545), mouse monoclonal antibodies to β-actin (cat. A5441), chicken polyclonal antibody to GFAP (cat. AB5541) were all from Sigma-Aldrich (St. Louis, MO, USA); mouse monoclonal antibody to PSD-95 (cat. 75028020, Antibodies Incorporated, Davis, CA, USA); rabbit monoclonal antibody to GFAP (cat. 12389, Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-Iba1 antibody (cat. 01919741, Wako, Richmond, VA, USA); rabbit monoclonal antibodies to synaptophysin (cat. ab32594), Olig2 (cat. ab136253), S100β (cat. ab52642) and CD11b (cat. ab133357) were from Abcam (Cambridge, MA, USA); mouse monoclonal antibody to NeuN (cat. mab377, Millipore, Temecula, CA, USA); Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 546 goat anti-mouse IgG, and Alexa Fluor 546 goat anti-rabbit IgG secondary antibodies were from Invitrogen (Carlsbad, CA, USA).

Techniques: Immunostaining, Staining, Control, Western Blot, Expressing, Isolation, Two Tailed Test

Journal: Cells

Article Title: Phagocytic Activities of Reactive Microglia and Astrocytes Associated with Prion Diseases Are Dysregulated in Opposite Directions

doi: 10.3390/cells10071728

Figure Lengend Snippet: Preparation of adult primary astrocyte cell cultures. ( A ) Co-immunostaining of PACs using an astrocyte-specific marker (GFAP, green) and microglia- (Iba1, red), oligodendrocyte- (olig2, red), neuron- (NeuN, red) or second astrocyte- (S100β, red) specific marker. Cell nuclei are stained with DAPI (blue). Images are representatives of three independent primary cell cultures, each prepared from an individual control animal. Scale bar = 50 μm. ( B ) Representative Western blots (left) and densitometric analysis of GFAP expression levels (right) in CT-PACs and 22L-PACs normalized per expression of β-actin. Data are expressed as mean ± SEM, n = 3 independent cultures isolated from individual animals, ** p < 0.01 (two-tailed, unpaired student t-test).

Article Snippet: The following antibodies were used: horseradish peroxidase (HRP), conjugated secondary anti-mouse IgG (cat. A9044), anti-rabbit IgG (cat. A0545), mouse monoclonal antibodies to β-actin (cat. A5441), chicken polyclonal antibody to GFAP (cat. AB5541) were all from Sigma-Aldrich (St. Louis, MO, USA); mouse monoclonal antibody to PSD-95 (cat. 75028020, Antibodies Incorporated, Davis, CA, USA); rabbit monoclonal antibody to GFAP (cat. 12389, Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal anti-Iba1 antibody (cat. 01919741, Wako, Richmond, VA, USA); rabbit monoclonal antibodies to synaptophysin (cat. ab32594), Olig2 (cat. ab136253), S100β (cat. ab52642) and CD11b (cat. ab133357) were from Abcam (Cambridge, MA, USA); mouse monoclonal antibody to NeuN (cat. mab377, Millipore, Temecula, CA, USA); Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 546 goat anti-mouse IgG, and Alexa Fluor 546 goat anti-rabbit IgG secondary antibodies were from Invitrogen (Carlsbad, CA, USA).

Techniques: Immunostaining, Marker, Staining, Control, Western Blot, Expressing, Isolation, Two Tailed Test