rabbit anti-post synaptic density protein 95 (psd95  (Thermo Fisher)

Journal: Heliyon

Article Title: Melatonin attenuates spatial learning and memory dysfunction in developing rats by suppressing isoflurane-induced endoplasmic reticulum stress via the SIRT1/Mfn2/PERK signaling pathway

doi: 10.1016/j.heliyon.2022.e10326

Figure Lengend Snippet: Melatonin mitigated isoflurane-induced hippocampal neuro apoptosis in neonatal rats. (A) Western blot bands of apoptosis signaling in the hippocampus. (B) The treatment of melatonin significantly reduced isoflurane induced increase of pro-apoptosis factor Bax expression in hippocampus of neonatal rats ( F (3, 20) = 71.360, P < 0.001). (C) The treatment of melatonin significantly reduced isoflurane induced decrease of anti-apoptosis factor Bcl-2 expression in hippocampus of neonatal rats ( F (3, 20) = 9.573, P < 0.001). (D) The treatment of melatonin significantly reduced isoflurane induced increase of pro-apoptosis factor cleaved-caspase 3 expression in hippocampus of neonatal rats ( F (3, 20) = 19.910, P < 0.001). (E) The treatment of melatonin significantly increase isoflurane induced decrease of synaptic protein PSD95 expression in hippocampus of neonatal rats ( F (3, 20) = 73.790, P < 0.001) (F) Immunofluorescence results of cleaved-caspase 3 and caspase 12 in hippocampus of all four groups rats. Expressions of cleaved-caspase 3 and caspase 12 in all four groups were presented through bar graphs: (G) for cleaved-caspase 3 ( F (3, 20) = 109.100, P < 0.001) and (H) for caspase 12 ( F (3, 20) = 105.200, P < 0.001). Data were presented as mean ± SEM (n = 6). ∗denotes p < 0.05, ∗∗denotes p < 0.01, ∗∗∗denotes p < 0.001 when comparing the two groups under each end of the capped line. Scale bar = 500 μm.

Article Snippet: The primary antibodies included the following: mouse anti-β-actin (1:2000; Qidongzi, Wuhan, China), mouse anti-SIRT1 (1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mitofusin 2 (Mfn2) (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-protein kinase RNA-like ER kinase (PERK) (1:500; Affinity Biosciences, OH, USA), rabbit anti- C/EBP homologues protein (CHOP) (1:500; Affinity Biosciences, OH, USA), rabbit anti-78 kDa glucose regulated protein (GRP78) (1:500; Affinity Biosciences, OH, USA), rabbit anti-activating transcription factor 4 (ATF4) (1:500; Affinity Biosciences, OH, USA), rabbit anti-cysteine containing aspartate specific protease 12 (caspase 12) (1:500; Proteintech North America, IL, USA), rabbit anti-Bcl-2-associated X protein (Bax) (1:500; Proteintech North America, IL, USA), rabbit anti-post-synaptic density protein 95 (PSD95) (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-B-cell lymphoma-2 (Bcl-2) (1:500; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-cleaved-cysteine containing aspartate specific protease 3 (cleaved-caspase 3).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Frontiers in Pediatrics

Article Title: Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure

doi: 10.3389/fped.2021.794544

Figure Lengend Snippet: (A) Diagram of mouse brain regions associated with mesolimbic dopamine pathway (PFC, prefrontal cortex; ACC, anterior cingulate cortex; NAC, nucleus accumbens; VTA, ventral tegmental area; Hippo, hippocampus; Amyg, amygdala). (B–D) Representative fluorescent confocal microscopic images of excitatory synaptic staining (presynaptic VGlut1, green; postsynaptic PSD95, red) at 10X and 63X magnification of the regions analyzed in this study: (B) ACC, (C) NAC, and (D) PFC.

Article Snippet: Guinea pig anti-vesicular glutamate transporter 1 (VGlut1; Cat. #AB5905, EMD Millipore, Burlington, MA) at 1:2,000 dilution was used to identify excitatory glutamatergic presynaptic axonal regions while rabbit anti-post synaptic density protein 95 (PSD95; Cat. #51-6900, Invitrogen, Carlsbad, CA) at 1:300 dilution was used to identify postsynaptic dendritic regions.

Techniques: Staining

Journal: Frontiers in Pediatrics

Article Title: Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure

doi: 10.3389/fped.2021.794544

Figure Lengend Snippet: Increased excitatory synapses with early life buprenorphine exposure. (A) Diagram illustrating co-localization of puncta representing presynaptic (VGlut1, green) and postsynaptic (PSD95, red) fluorescent antibody label pairs for quantifying excitatory glutamatergic synapses. (B–D) Representative IHC images (left) and quantification (right) of co-localized VGlut1 (green) and PSD95 (red) excitatory synaptic puncta (yellow arrowheads) from prenatal drug-exposed WT C57Bl/6J P21 mouse brain within (B) the anterior cingulate cortex (ACC), (C) nucleus accumbens (NAC), and (D) prefrontal cortex (PFC). n = 7 (vehicle control), 6 (GBP), 7 (Bup+GBP), 6 (Bup); ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Guinea pig anti-vesicular glutamate transporter 1 (VGlut1; Cat. #AB5905, EMD Millipore, Burlington, MA) at 1:2,000 dilution was used to identify excitatory glutamatergic presynaptic axonal regions while rabbit anti-post synaptic density protein 95 (PSD95; Cat. #51-6900, Invitrogen, Carlsbad, CA) at 1:300 dilution was used to identify postsynaptic dendritic regions.

Techniques:

Journal: Frontiers in Pediatrics

Article Title: Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure

doi: 10.3389/fped.2021.794544

Figure Lengend Snippet: Dual exposure to buprenorphine and gabapentin decreased inhibitory synapses at P21. (A) Diagram illustrating co-localization of puncta representing presynaptic (VGAT, green) and postsynaptic (PSD95) fluorescent antibody label pairs for quantifying inhibitory GABAergic synapses. (B–D) Representative IHC images (left) and quantification (right) of co-localized VGAT (green) and gephyrin (red) inhibitory synaptic puncta (yellow arrowheads) from prenatal drug-exposed WT C57Bl/6J P21 mouse brain within (B) ACC, (C) NAC, and (D) PFC. n = 7 (vehicle control), 6 (GBP), 7 (Bup+GBP), 6 (Bup); ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Guinea pig anti-vesicular glutamate transporter 1 (VGlut1; Cat. #AB5905, EMD Millipore, Burlington, MA) at 1:2,000 dilution was used to identify excitatory glutamatergic presynaptic axonal regions while rabbit anti-post synaptic density protein 95 (PSD95; Cat. #51-6900, Invitrogen, Carlsbad, CA) at 1:300 dilution was used to identify postsynaptic dendritic regions.

Techniques:

Journal: Frontiers in Pediatrics

Article Title: Alterations in Excitatory and Inhibitory Synaptic Development Within the Mesolimbic Dopamine Pathway in a Mouse Model of Prenatal Drug Exposure

doi: 10.3389/fped.2021.794544

Figure Lengend Snippet: Altered excitatory synaptic connectivity following prenatal drug treatment in α2δ-1 haploinsufficient mice. Representative IHC images (left) of co-localized VGlut1 (green) and PSD95 (red) excitatory synaptic puncta (yellow arrowheads) from prenatal drug-exposed α2δ-1 +/- (Het) C57Bl/6J P21 mouse brain with quantification (right) compared to WT C57Bl/6J P21 mouse brains with the same prenatal treatment within (A) ACC, (B) NAC, and (C) PFC. Het: n = 11 (vehicle control), 7 (GBP), 7 (Bup+GBP), 7 (Bup); * p < 0.05; **** p < 0.0001.

Article Snippet: Guinea pig anti-vesicular glutamate transporter 1 (VGlut1; Cat. #AB5905, EMD Millipore, Burlington, MA) at 1:2,000 dilution was used to identify excitatory glutamatergic presynaptic axonal regions while rabbit anti-post synaptic density protein 95 (PSD95; Cat. #51-6900, Invitrogen, Carlsbad, CA) at 1:300 dilution was used to identify postsynaptic dendritic regions.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Histological Correlates of Neuroanatomical Changes in a Rat Model of Levodopa-Induced Dyskinesia Based on Voxel-Based Morphometry

doi: 10.3389/fnagi.2021.759934

Figure Lengend Snippet: L-DOPA treatment significantly increased the levels of GFAP and synaptic-associated proteins (SYP, PSD95, SAP97), but not vasculature, in the lesioned striatum. (A) Representative western blots of TH, GFAP, VEGF, and synaptic associated proteins in the lesioned striatum of the three groups. GAPDH served as the internal control. (B–G) Quantitative analysis of the aforementioned proteins. Data were presented as% of the sham group. * P < 0.05, ** P < 0.01, **** P < 0.0001 vs. the specified or sham group; ## P < 0.01 vs. the PD group. All results were presented as mean ± SEM ( N = 4/group).

Article Snippet: After washing, the membranes were incubated using the following primary antibodies overnight at 4°C: anti-TH Rabbit pAb (Tyrosine hydroxylase, 1:3000, Proteintech, 25859-1-AP); anti-GFAP Mouse mAb (glial fibrillary acidic protein, 1:2500, Servicebio, GB12096); anti-VEGF Mouse mAb (vascular endothelial growth factor, 1:200, Santa Cruz, sc-7269); anti-SYP Rabbit pAb (synaptophysin, 1:10000, Servicebio, GB11553); anti-PSD95 Rabbit pAb (post synaptic density protein 95, 1:500, Servicebio, GB11277); anti-SAP97 Rabbit pAb (synapse-associated protein 97, 1:2000, Abcam, ab3437); anti-GAPDH Rabbit pAb (glyceraldehyde 3-phosphate dehydrogenase, 1:10000, GeneTex, GTX100118).

Techniques: Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Histological Correlates of Neuroanatomical Changes in a Rat Model of Levodopa-Induced Dyskinesia Based on Voxel-Based Morphometry

doi: 10.3389/fnagi.2021.759934

Figure Lengend Snippet: L-DOPA treatment promoted astrocytic and synaptic pathology in the lesioned striatum. (A) TH immunostaining of the striatum in each group. (C,E,G,I) Representative immunohistochemical images of GFAP, VEGF, and synaptic associated proteins (SYP, PSD95) of the dorsolateral striatum from the three groups in the lesioned side. [Scale bars: (A) is 1 mm, (C,E,G,I) is 50 μm]. (B,D,F,H,J) Analysis of differences of the aforementioned proteins. AOD, average optical density. Results were expressed as % of the unlesioned side. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the specified group. Error bars represent SEM ( N = 3/group).

Article Snippet: After washing, the membranes were incubated using the following primary antibodies overnight at 4°C: anti-TH Rabbit pAb (Tyrosine hydroxylase, 1:3000, Proteintech, 25859-1-AP); anti-GFAP Mouse mAb (glial fibrillary acidic protein, 1:2500, Servicebio, GB12096); anti-VEGF Mouse mAb (vascular endothelial growth factor, 1:200, Santa Cruz, sc-7269); anti-SYP Rabbit pAb (synaptophysin, 1:10000, Servicebio, GB11553); anti-PSD95 Rabbit pAb (post synaptic density protein 95, 1:500, Servicebio, GB11277); anti-SAP97 Rabbit pAb (synapse-associated protein 97, 1:2000, Abcam, ab3437); anti-GAPDH Rabbit pAb (glyceraldehyde 3-phosphate dehydrogenase, 1:10000, GeneTex, GTX100118).

Techniques: Immunostaining, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways

doi: 10.3390/ijms21155239

Figure Lengend Snippet: Select pro-apoptotic members of the Bcl-2 family are upregulated in irradiated primary cortical neurons. Post-ChIP qPCR to examine occupancy of p53 on Noxa promoter region ( A ). (T(4) = 2.08, p = 0.0001). Data presented as fold change to non-irradiated control group. n = 3/group. Significance assigned based on one-tailed t-test, *** p < 0.001 versus control Rat primary cortical neurons (RCNs). qPCR quantification of Noxa ( B ) (F(9,40) = 63.76, p < 0.0001 for 24 h 2Gy and for 6 h and 24 h 8 and 32Gy, compared to control), Puma ( C ) (F(9,40) = 56.47, p = 0.0051 for 6 h 2Gy, p = 0.0002 for 30 min 32Gy, and p < 0.0001 for 6 and 24 h 8Gy and 32Gy, compared to control), Bim ( D ) (F(9,40) = 65.12, p = 0.0044 for 6 h 2Gy, p < 0.0001 for all-time points treated with 8 and 32Gy except 30 min 8Gy, compared to control). Data presented as fold change to non-irradiated control group. n = 5 + /group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. Irradiation induces dose-dependent neuronal cell death and time- and dose-dependent activation of caspase-dependent apoptosis. Irradiation induces dose-dependent neuronal cell death, and time- and dose-dependent activation of caspase-dependent apoptosis in RCNs. Neurons were irradiated with 2, 8 and 32Gy. Twenty-four hours later, LDH release was measured ( E ) (F(3,36)= 7.43, p = 0.0175 for 2Gy, p < 0.0001 for 8 and 32Gy). Data expressed as a percentage of levels of non-irradiated control. n = 10/group, * p < 0.05, **** p < 0.0001 vs. control. Neurons were collected 30 min and 24 h after 2, 8 and 32Gy IR. Whole-cell lysates were separated by SDS-polyacrylamide gel and immunoblotted with antibodies against cleaved caspase-3, PARP, α-fodrin, PSD95 and β-actin ( F ). ( G ) Quantification of levels of cleaved caspase 3 (F(6,14) = 248.2, p < 0.0001 for 24 h at all doses, compared to control), PARP (F(6,14) = 79.08, p < 0.0001 for 24 h at 8 and 32Gy, compared to control), α-fodrin (120kDa) (F(6,14) = 553.4, p < 0.0001 for 24 h at all doses, compared to control) and PSD95 ( H ) (F(6,14) = 35.55, p = 0.0087 for 30 min 2Gy, p < 0.0001 at all other doses/times, except 30 min 32Gy, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001 vs. control.

Article Snippet: The following antibodies were used in this study: Histone H2A.X (ab11175; Abcam); AIF (sc-13116), cytochrome c (sc-13560; Santa Cruz Biotechnology, Dallas, TX, USA); ((Ph-H2A.X(Ser139)-γ-H2A.X) (#9718), phosphorylated ataxia telangiectasia mutated kinase ((Ph-ATM (Ser1981)-Ph-ATM), (Phospho-ATR(Ser428)-Ph-ATR) (#2853), Cleaved Caspase-3 (#9661), PARP (#9542), (phospho-p53(Ser15)-Ph-p53) (#9284), PUMA (#14570), p53 (#2524), post-synaptic density protein 95 (PSD95) (#3450) (Cell Signaling); GAPDH (ADI-CSA-335) and α-fodrin (BML-FG6090; Enzo Life Sciences, Inc., Farmingdale, NY, USA); β-actin (A1978; Sigma, St. Louis, MO); phospho-ATM (05–740 Millipore, Burlington, MA, USA); p21 (556430 BD Biosciences, San Jose, CA, USA).

Techniques: Irradiation, ChIP-qPCR, Control, One-tailed Test, Activation Assay