Cultured cells were fixed with 4% PFA for 10 min and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After, the cells were blocked with 3% bovine serum albumin and 5% normal goat serum (Sigma, St. Louis, MO, USA) in PBS (blocking solution) for 1 h and, then, incubated overnight at 4°C with the specified primary antibodies diluted in blocking solution. The primary antibodies were mouse anti-β-tubulin III (Promega, Madison, WI, USA; 1:1000), rabbit anti-cleaved caspase-3 (Cell Signaling, Beverly, MA, USA; 1:50), rabbit anti-GFAP (Dako Corporation, Glostrup, Denmark; 1:1000), mouse anti-synaptophysin (Chemicon International, Billerica, MA, USA; 1:1000), rabbit anti-PSD-95 (Cell Signaling Technology, Beverly, MA, USA; 1:100), mouse anti-SMADs 2/3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200). After primary antibody incubation, the cells were extensively washed with PBS and incubated with secondary antibodies for 2 h at room temperature. Secondary antibodies were Alexa Fluor 546 (goat anti-rabbit IgG, goat anti-mouse IgG; Molecular Probes, Paisley, UK; 1:1000) or Alexa Fluor 488 (goat anti-rabbit IgG, goat anti-guinea pig IgG, goat anti-mouse IgG; Molecular Probes, Paisley, UK; 1:300). Nuclei were counterstained with DAPI (Sigma Chemical Co., St. Louis, MO, USA). The cells were observed with the aid of a TE2000 Nikon microscope.
Rabbit anti psd95
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-PSD95 is a primary antibody that recognizes the PSD95 (postsynaptic density protein 95) protein. PSD95 is a scaffold protein found in the postsynaptic density of excitatory synapses and is involved in the organization and regulation of the postsynaptic signaling complex.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Mouse anti map2, by Merck Group (3 mentions)
Mouse anti β actin, by Merck Group (3 mentions)
Mouse anti synaptophysin, by Merck Group (2 mentions)
Mouse anti rac1, by Merck Group (2 mentions)
Rabbit anti glur1, by Cell Signaling Technology (2 mentions)
Rabbit anti synapsin 1, by Cell Signaling Technology (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Rabbit anti synaptophysin, by Proteintech (2 mentions)
Rabbit anti synaptophysin, by Cell Signaling Technology (2 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Mouse anti β tubulin 3, by Promega (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti rhoa, by Cytoskeleton (1 mentions)
Helios gene gun system, by Bio-Rad (1 mentions)
28 protocols using rabbit anti psd95
1
Immunofluorescence Staining of Cultured Cells
2
Detecting Neuronal Cytoskeletal Dynamics
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For Western immunoblotting analysis, infected neurons were collected in 1 × LDS loading buffer (Invitrogen) and boiled for 10 min. Proteins were separated by electrophoresis in 12% NuPAGE gels (Invitrogen). To better detect ATF4, the gels were run a longer time (80–100 min) to separate ATF4 from a closely migrating non-specific band. The following primary antibodies were used: rabbit anti-ATF4 (1:250, PRF&L), mouse anti-RhoA (1:1000; Cytoskeleton), mouse anti-RhoA-GTP (1:500; Santa Cruz), mouse anti-Rac1 (1:5000; Millipore), mouse anti-Cdc42 (1:500; BD), rabbit anti-pLIMK1/2 (1:500; Santa Cruz), and mouse anti-GAPDH (1:2000; Imgenex).
Immunocytochemistry was performed on the primary hippocampal neurons cultured on 15-mm cover glasses. Neurons were fixed with 4% PFA for 15 min and blocked with 5% BSA. The following primary antibodies were used: rabbit anti-GluR1 (1:200; Cell Signaling), rabbit anti-PSD-95 (1:200; Cell Signaling).
For DiOlistic labeling, the Helios gene gun system (Bio-Rad) was used according to the manufacturer's instructions. Tungsten particles (1.1 μm, Bio-Rad) coated with Dil (Invitrogen), which defines the neuronal architecture in red, were delivered into fixed neurons on coverslips or brain sections. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and imaged the next day.
Immunocytochemistry was performed on the primary hippocampal neurons cultured on 15-mm cover glasses. Neurons were fixed with 4% PFA for 15 min and blocked with 5% BSA. The following primary antibodies were used: rabbit anti-GluR1 (1:200; Cell Signaling), rabbit anti-PSD-95 (1:200; Cell Signaling).
For DiOlistic labeling, the Helios gene gun system (Bio-Rad) was used according to the manufacturer's instructions. Tungsten particles (1.1 μm, Bio-Rad) coated with Dil (Invitrogen), which defines the neuronal architecture in red, were delivered into fixed neurons on coverslips or brain sections. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and imaged the next day.
3
Immunoblotting Analysis of Synaptic Proteins
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Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. DL-AP5 (0105), CNQX (0190) were purchased from Tocris Bioscience. Following antibodies were used: Mouse anti-Nrg1 (Santa Cruz Biotechnology) (sc-393006; 1:1000 for blotting); Mouse anti-ErbB4 (Santa Cruz Biotechnology) (sc-8050; 1:1000 for blotting); Rabbit anti-P-ErbB4 (Cell Signaling Technology) (Tyr1284; 1:1000 for blotting); Mouse anti-GAPDH (Santa Cruz Biotechnology) (sc-32233; 1:1000 for blotting); Rabbit anti-PSD95 (Cell Signaling Technology) (#3450; 1:1000 for blotting); Mouse anti-Gephyrin (Santa Cruz Biotechnology) (sc-25311; 1:1000 for blotting); Rabbit anti-Synaptotagmin-1 (Cell Signaling Technology) (#14,558; 1:1000 for blotting); Rabbit anti-GABARa1 (Millipore) (3,108,661; 1:1000 for blotting); Rabbit anti-GluN2B(NMDAR2B) (Cell Signaling Technology) (#14,544; 1:1000 for blotting); Rabbit anti-GluN2A (Cell Signaling Technology) (#4205; 1:1000 for blotting); Rabbit anti- GluR1 (Cell Signaling Technology) (#13,185; 1:1000 for blotting).
4
Immunohistochemical Analysis of Synaptic Markers
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Rats were anesthetized completely with sodium pentobarbital (50 mg/kg) and perfused intracardially with saline followed by 4% paraformaldehyde. Ipsilateral brain tissue was removed and rapidly immerged in 4% paraformaldehyde. Sections (10 μm thickness) were immersed in 3% hydrogen peroxide to block endogenous peroxidase. After washing thoroughly three times with PBS, immunostaining was performed with the following primary antibodies: rabbit anti-SYN (1:1000; Proteintech, Chicago, IL, USA), rabbit anti-synapsin I (1:1000; Cell Signaling Technology, Boston, MA, USA), and rabbit anti-PSD 95 (1:1000; Cell Signaling Technology) at 4°C overnight. Sections were then washed followed by incubation with biotinylated-conjugated anti-rabbit secondary antibody (1:800; Proteintech) for 2 hours at room temperature. Last, sections were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin. Images were measured using Image-Pro-Plus (Media Cybernetics, Rockville, MD, USA). Three random fields per section were selected from each animal, and the optical density ratio was calculated: this included the optical density of the region and background from each section.
5
Antibody Characterization for Neuronal Imaging
6
Quantifying Neuronal Synaptic Density in TBI
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Mice were decapitated, then brain was harvested on day 25 after TBI (Figure S2A ). Immunofluorescence staining was conducted as previously described.15 In brief, the brain tissues were fixed in 4% paraformaldehyde at 4°C for 48 h and dehydrated in ethanol and embedded in paraffin. The coronal mouse brain sections were obtained. After dewaxing and rehydration, the sections were treated with 0.01 M citrate buffer (pH 6.0) with 0.1% Tween‐20 at 90–95°C for 5 min for antigen retrieval. The sections were incubated at 4 °C overnight with the mouse anti‐NeuN (1:200; Millipore) and rabbit anti‐PSD95 (1:200; Cell Signaling, Inc.). After washing with PBS, the sections were stained with the secondary antibody, anti‐mouse IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 488 Conjugate) (1:1000; Cell Signaling, Inc.) and anti‐rabbit IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 594 Conjugate) (1:1000, Cell Signaling, Inc.) for 1 h in the dark at room temperature, and then stained with DAPI (Beyotime Institute of Biotechnology) for 1 min to reveal the nuclei, and analyzed using a laser scanning confocal microscope (Leica). The images were taken using a laser scanning confocal microscope (Leica).
7
Synaptic Protein Expression Analysis
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Ipsilateral brain tissue samples were homogenized in radioimmunoprecipitation assay lysis buffer, and centrifuged at 12,000 r/min for 5 minutes at 4°C. Supernatants were collected. Protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein (30 μg per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. Membranes were incubated with rabbit anti-synaptophysin (1:500; Proteintech), rabbit anti-PSD 95 (1:1000; Cell Signaling), and rabbit anti-synapsin I (1:1000; Cell Signaling) overnight at 4°C. After washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000; Proteintech) for 50 minutes at room temperature. Enhanced chemiluminescence solution (Thermo Fisher Scientific) was used for detecting immunoreactive bands. Last, membranes were developed using a Bio-Rad ChemiDoc XRS digital documentation system (Bio-Rad). Synapsin band densities were quantified and normalized using mouse anti-β-actin (1:4000). Results are presented by averaging the values (protein/β-actin).
8
Western Blotting Analysis of Neuroinflammatory Markers
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As previously described by us,
40 (link) proteins were extracted from the MCA‐supplied brain regions and loaded onto SDS‐polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti‐NLRP3, Merck; 1:500, rabbit anti‐ASC, Abcam; 1:1000, rabbit anti‐IL‐1β, Abcam; 1:1000, rabbit anti‐IL‐18, Proteintech; 1:1000, rabbit anti‐caspase‐1, Abcam; 1:500, rabbit anti‐GSDMD, Affinity; 1:2000, rabbit anti‐G3BP1, Proteintech; 1:500, rabbit anti‐TIA1, Proteintech; 1:1000, rabbit anti‐DDX3X, Proteintech; 1:1000, rabbit anti‐IgG antibody, Cell Signaling Technology; 1:10,000, rabbit anti‐SYN, Abcam; 1:1000, rabbit anti‐PSD‐95, Cell Signaling Technology; 1:1000, rabbit anti‐BDNF, Abcam; 1:500, rabbit anti‐Ang‐1, Proteintech; 1:500, rabbit anti‐Ang‐2, Proteintech; 1:500, rabbit anti‐VEGF, Abcam; 1:1000, mouse β‐actin, Santa Cruz) for 24 h at 4°C. Next, membranes were incubated with a secondary antibody (1:4000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Cell Signaling Technology) for 2 h at room temperature. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health) to quantify protein expression in terms of relative image density.
40 (link) proteins were extracted from the MCA‐supplied brain regions and loaded onto SDS‐polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti‐NLRP3, Merck; 1:500, rabbit anti‐ASC, Abcam; 1:1000, rabbit anti‐IL‐1β, Abcam; 1:1000, rabbit anti‐IL‐18, Proteintech; 1:1000, rabbit anti‐caspase‐1, Abcam; 1:500, rabbit anti‐GSDMD, Affinity; 1:2000, rabbit anti‐G3BP1, Proteintech; 1:500, rabbit anti‐TIA1, Proteintech; 1:1000, rabbit anti‐DDX3X, Proteintech; 1:1000, rabbit anti‐IgG antibody, Cell Signaling Technology; 1:10,000, rabbit anti‐SYN, Abcam; 1:1000, rabbit anti‐PSD‐95, Cell Signaling Technology; 1:1000, rabbit anti‐BDNF, Abcam; 1:500, rabbit anti‐Ang‐1, Proteintech; 1:500, rabbit anti‐Ang‐2, Proteintech; 1:500, rabbit anti‐VEGF, Abcam; 1:1000, mouse β‐actin, Santa Cruz) for 24 h at 4°C. Next, membranes were incubated with a secondary antibody (1:4000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Cell Signaling Technology) for 2 h at room temperature. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health) to quantify protein expression in terms of relative image density.
9
Detecting Newly Synthesized Proteins by PLA
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Detection of newly synthesized proteins by PLA was performed as previously described (5 (link), 10 (link), 45 (link)). Immunostaining using mouse antipuromycin (Kerafast; 1:500) antibody in combination with rabbit anti–β-actin (Abcam; 1:1,000), rabbit anti–PSD-95 (cell signaling technologies; 1:1,000), or rabbit anti-Camk2a (Thermo; 1:1,000) was performed overnight at 4 °C. Following 5× PBS washes, PLA was performed (Sigma). Rabbit PLAplus and mouse PLAminus probes were used. PLA was performed according to the manufacture’s guidelines. Following PLA, anti-Map2 immunostaining (guinea pig anti-Map2, Cell Signaling; 1:5,000) was performed to label dendrites. Samples were imaged using a 40× oil objective on a LSM780 or LSM880. Z-stacks (0.43 μm) spanned the entire volume of imaged neurons. Images were analyzed using ImageJ. A 100-μm segment of the dendrite was assessed for the number of Puro-PLA puncta and the density of signal was calculated.
10
Immunofluorescence Imaging of Hippocampal Slices
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Hippocampal 450 μm-thick transversely coronal slices were treated or not with sCT PFOs- or Monomer-enriched solutions for 100 min. After treatment, the slices were fixed over night with 4% paraformaldehyde in PBS, 0.12 M in sucrose. After fixation, the samples were rinsed three times in PBS with 5% sucrose and 0.15 mM CaCl2 and left overnight in sucrose buffer (PBS with 30% sucrose and 0.15 mM CaCl2). Samples were then embedded in Tissue Freezing Medium (Jung, Germany), frozen at −30 °C in isopentane and stored at −80 °C. Sections (8 μm) were cut at a Leica CM 1860 UV cryostat and labelled with primary antibodies overnight at 4 °C. The following primary antibodies were used: monoclonal anti-NeuN (Millipore, USA) rabbit anti-PSD-95 (Cell Signaling Technology, Danvers, MA) and monoclonal anti-synaptophysin (BD Transduction Laboratories, Franklin Lakes, NJ). Primary antibodies were revealed with secondary antibodies coupled to Alexa Fluor® 488 and Alexa Fluor® 546 (Invitrogen), diluted 1:250 PBS (45 min, 37 °C). Sections were counterstained with Hoechst 33258; the dye, which binds specifically to A–T base regions in DNA and emits blue immunofluorescence at 350 nm, was administered at 1 ng/ml for 1 minute. Sections were observed at an Eclipse 80i Nikon Fluorescence Microscope (Nikon Instruments, Amsterdam, The Netherlands), equipped with a Video Confocal (ViCo) system.
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