Journal: Brain research bulletin

Article Title: PSD-95 inhibitor Tat-NR2B9c (NA-1) protects the integrity of the blood-brain barrier after transient middle artery occlusion in rats by downregulating matrix metalloprotease-9 and upregulating endothelial nitric oxide synthase.

doi: 10.1016/j.brainresbull.2023.110836

Figure Lengend Snippet: Fig. 4. PSD-95 is expressed in neurons and vascular endothelial cells in the brain. (A-B) Representative micrographs of cerebral ischemic penumbra ipsilateral and contralateral after immunostaining against PSD-95 and (A) the neuronal marker NeuN or (B) the vascular endothelial cell marker PECAM-1. (C-D) Quantitation of the immunostaining experiments in panels A-B. Data are mean ± SEM for five animals per condition. * P < 0.05, * * P < 0.01, * ** P < 0.001, * ** * P < 0.0001.

Article Snippet: Subsequently, they were incubated overnight at 4 ◦C with antibodies against the following proteins: PSD-95 (1:100; catalog no. AB238135, Abcam), nNOS (1:100; catalog no. AB76067, Abcam), eNOS (1:100; catalog no. AB76067, Abcam), PECAM-1 (1:800; catalog no. SC5678, Santa Cruz, Dallas, TX, USA), and NeuN (1:400; catalog no. 66836–1-Ig, Proteintech).

Techniques: Immunostaining, Marker, Quantitation Assay

Journal: Cell death & disease

Article Title: ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis.

doi: 10.1038/s41419-023-06237-6

Figure Lengend Snippet: Fig. 3 Effects of ChemR23 activation with RvE1 or C-9 on neuronal damage and synaptic dysfunction in CCH rats. A Representative H&E staining of the rat hippocampus. Scale bar: 200 μm (upper panel) or 50 μm (lower panel). n = 3 per group. B Representative Nissl staining in the rat hippocampus. Scale bar: 50 µm. n = 3 per group. C Golgi staining in the rat hippocampus. Scale bar: 200 μm. n = 3 per group. D, E Enlarged images of neuronal dendrites by Golgi staining and quantification of spine density. n = 3 per group. F Quantification of Nissl- positive cells in the rat hippocampus. n = 3 per group. G–I Representative immunoblotting bands and semiquantitative analysis of SYN and PSD95 in the rat hippocampus. n = 4 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CCH.

Article Snippet: For in vivo fluorescence staining, after deparaffinization and antigen retrieval (0.05% citraconic acid), the paraffin-embedded sections were treated with endogenous peroxidase (3% H2O2 in PBS) for 10min, followed by incubation with 5% donkey serum containing 0.1% Triton X-100 in PBS for 1 h. Next, the sections were incubated overnight at 4 °C with the following primary antibodies: CMKLR1 (1:200, No. 10325, Cayman, Michigan, USA), NLRP3 (1:500, BA3677, Boster, Wuhan, China), GSDMD (1:500, A22523, ABclonal, Wuhan, China), PSD95 (1:100, 17785-1-AP, Proteintech, Wuhan, China), SYN (1:400, 17785-1-AP, Proteintech, Wuhan, China), GFAP (1:200, 60190-1-Ig, Proteintech, Wuhan, China), Iba-1 (1:500, 66827-1-Ig, Proteintech, Wuhan, China) and NeuN (1:100, 66836-1-Ig, Proteintech, Wuhan, China), followed by incubation with the corresponding fluorescent secondary antibody the next day.

Techniques: Activation Assay, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Clec7a Signaling in Microglia Promotes Synapse Loss Associated with Tauopathy

doi: 10.3390/ijms26072888

Figure Lengend Snippet: Synapse loss and microglia activation in the hippocampus of PS19 mice. ( A ) Synaptic proteins extracted from the hippocampus of 6-month-old wild-type (WT) and PS19 mice were analyzed via Western blotting. ( B , C ) Quantitative analysis of PSD95 ( B ) and synaptophysin ( C ) expression levels ( n = 6 mice per group, unpaired Student’s t -test). ( D ) Representative single-plane images depicting the localization of PSD95 (green) and synaptophysin (Syp) (red) in the hippocampus of 6-month-old WT and PS19 mice. Dotted circles highlight colocalized synapses. Scale bars, 5 μm. ( E – G ) Quantification of synaptic density ( n = 6 mice per group, unpaired Student’s t -test). ( H ) Immunofluorescence images showing CD68+ lysosome content (green) within Iba1 + microglia (red) in the hippocampus of 6-month-old WT and PS19 mice. Scale bars, 50 μm. ( I ) Quantification of the number of Iba1 + microglial cells ( n = 6 mice per group, unpaired Student’s t -test). ( J ) Quantification of CD68+ lysosome volume ( n = 6 mice per group, unpaired Student’s t -test). * p < 0.05; ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD.

Article Snippet: The primary antibodies used in this study included Clec7a (1:200, AF1756-SP, R&D, Minneapolis, MN, USA); MD2(1:200, GTX85517, GeneTex, San Antonio, TX, USA); Iba-1 (1:1000, ab178846, Abcam, Cambridge, MA, USA); Iba-1 (1:500, ab283346, Abcam, Cambridge, MA, USA); NueN (1:500, 66836-1-lg, Proteintech, Wuhan, China); GFAP (1:500, 3670, CST, Danvers, MA, USA); synaptophysin (1:200, ab16659, abcam, Cambridge, MA, USA); PSD95 (1:200, MAB1596, Millipore Sigma, Burlington, MA, USA); and CD68 (1:200, MCA1957, Bio-RAD, Inc., Hercules, CA, USA), followed by incubation with secondary antibodies (1:1000, CST, Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Clec7a Signaling in Microglia Promotes Synapse Loss Associated with Tauopathy

doi: 10.3390/ijms26072888

Figure Lengend Snippet: Clec7a is specifically increased in the hippocampus of PS19 mice and closely associated with synaptic loss. ( A – D ) Representative immunoblots of Clec7a and P-SYK in ventral hippocampal tissue from WT or PS19 mice at 3 months ( A ), 6 months ( B ), 9 months ( C ), and 12 months ( D ) of age. ( E , F ) Quantification of Clec7a and P-SYK levels shown in panel ( A – D ) ( n = 6 mice per group, unpaired Student’s t test). ( G – I ) Representative confocal images of Clec7a (red) and Iba1 + microglia (green) in the whole brain of WT and PS19 mice at 3 months ( G ), 6 months ( H ), and 9 months ( I ). Scale bars, 1 mm. ( J ) Representative images showing Iba1 (green) and Clec7a (red) staining in the dentate gyrus (DG) regions of hippocampal tissue from WT and PS19 mice at 6 months. Scale bars, 50 μm. ( K , L ) Quantitative analysis of Clec7a intensity and the number of Clec7a-positive Iba1 ( n = 3 mice per group, unpaired Student’s t test). ( M ) Confocal images of synaptic puncta engulfed by microglia showing the colocalization of PSD95 with CD68 within microglia in the DG regions of hippocampal tissue from WT and PS19 mice at 6 months. Scale bars, 5 μm. ( N ) Quantitative analysis of ( M ) ( n = 6 mice per group, unpaired Student’s t test). ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD.

Article Snippet: The primary antibodies used in this study included Clec7a (1:200, AF1756-SP, R&D, Minneapolis, MN, USA); MD2(1:200, GTX85517, GeneTex, San Antonio, TX, USA); Iba-1 (1:1000, ab178846, Abcam, Cambridge, MA, USA); Iba-1 (1:500, ab283346, Abcam, Cambridge, MA, USA); NueN (1:500, 66836-1-lg, Proteintech, Wuhan, China); GFAP (1:500, 3670, CST, Danvers, MA, USA); synaptophysin (1:200, ab16659, abcam, Cambridge, MA, USA); PSD95 (1:200, MAB1596, Millipore Sigma, Burlington, MA, USA); and CD68 (1:200, MCA1957, Bio-RAD, Inc., Hercules, CA, USA), followed by incubation with secondary antibodies (1:1000, CST, Danvers, MA, USA).

Techniques: Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Clec7a Signaling in Microglia Promotes Synapse Loss Associated with Tauopathy

doi: 10.3390/ijms26072888

Figure Lengend Snippet: Inhibition of Clec7a effectively mitigates microglial synaptic phagocytosis and neuroinflammation in PS19 mice. ( A ) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-month-old WT mice and Laminarin-administered PS19 mice. ( B ) Quantitation of Clec7a, P-SYK, NLRP3, ASC, Procaspase-1, and IL-1β proteins in ( A ) ( n = 6 mice per group, unpaired Student’s t test). ( C ) Representative Western blots of indicated proteins in the ventral hippocampal lysates from 6-months old WT mice and Laminarin-administered PS19 mice. ( D , E ) Quantitation of PSD95 ( D ) and synaptophysin ( E ) proteins ( n = 6 mice per group, unpaired Student’s t test). ( F ) Confocal images of synaptic puncta engulfed by microglia showing the colocalization of PSD95 (green) with CD68 (grays) within Iba1 + microglia (red) in the DG regions of hippocampal tissue from WT mice and Laminarin-administered PS19 mice. Scale bars, 5 μm. ( G ) Quantitation of CD68 volume. ( H ) Quantitation of PSD95 engulfment within CD68+ microglial structures ( n = 6 mice per group, one-way ANOVA test). * p < 0.05; ** p < 0.01; *** p < 0.001. Data are presented as mean ± SD.

Article Snippet: The primary antibodies used in this study included Clec7a (1:200, AF1756-SP, R&D, Minneapolis, MN, USA); MD2(1:200, GTX85517, GeneTex, San Antonio, TX, USA); Iba-1 (1:1000, ab178846, Abcam, Cambridge, MA, USA); Iba-1 (1:500, ab283346, Abcam, Cambridge, MA, USA); NueN (1:500, 66836-1-lg, Proteintech, Wuhan, China); GFAP (1:500, 3670, CST, Danvers, MA, USA); synaptophysin (1:200, ab16659, abcam, Cambridge, MA, USA); PSD95 (1:200, MAB1596, Millipore Sigma, Burlington, MA, USA); and CD68 (1:200, MCA1957, Bio-RAD, Inc., Hercules, CA, USA), followed by incubation with secondary antibodies (1:1000, CST, Danvers, MA, USA).

Techniques: Inhibition, Western Blot, Quantitation Assay