Mice brain tissues were fixed with a 10% formalin solution for 48–72 h and then dehydrated and embedded into paraffin and cut into 4 µm coronary sections. Every three hippocampal slices with similar anatomical regions for each mouse were stained with each antibody. The slices were dewaxed, hydrated by gradient alcohol, and then repaired with a sodium citrate buffer and blocked with normal goat serum sealing fluid (ZSGB-BIO, Beijing, China). These were incubated with anti-Aβ17-24 antibody (1:300, 4G8, BioLegend, San Diego, CA, USA), anti-PSD-95 antibody (1:300, Abcam, Cambridge, UK), anti-MAP2 antibody (1:300, abcam), or anti-vimentin antibody (1:100, CST, Framingham, MA, USA) overnight at 4 °C. HRP-labeled anti-rabbit/mouse IgG (ZSGB-BIO) was used to identify antigens, and DAB (ZSGB-BIO) was performed to stain. The slices were collected in images by Olympus cellSens (Tokyo, Japan) software and analyzed by image J (NIH) software (Media Cybernetics Inc., Rockville, MD, USA).
Anti psd 95 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-PSD-95 antibody is a protein-specific antibody that binds to the PSD-95 (postsynaptic density protein 95) protein. PSD-95 is a key structural and functional component of the postsynaptic density in excitatory synapses. This antibody can be used to detect and study the localization and expression of PSD-95 in various biological samples.
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Lab products found in correlation
Anti map2 antibody, by Abcam (2 mentions)
Anti syn antibody, by Abcam (2 mentions)
Rabbit anti bdnf antibody, by Abcam (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Nih image j software, by Media Cybernetics (1 mentions)
Cellsens, by Olympus (1 mentions)
Irdye 680rd donkey anti mouse igg, by LI COR (1 mentions)
Phospho akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Akt pan 11e7 rabbit mab, by Cell Signaling Technology (1 mentions)
Phospho p70 s6 kinase thr421 ser424, by Cell Signaling Technology (1 mentions)
Ube3a, by Abcepta (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
β actin antibody, by Merck Group (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by ZSGB-BIO (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by LI COR (1 mentions)
Citric acid, by Solarbio (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat serum, by Beyotime (1 mentions)
Cy3 conjugated anti rabbit igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
10 protocols using anti psd 95 antibody
1
Immunohistochemical Analysis of Alzheimer's Markers
2
Protein Expression Profiling Assay
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NuPAGE Novex Midi Gels (4–12%) from Life Technologies were used for protein separation. The following antibodies were used: Anti-PSD95 antibody (Abcam, Cat. #ab18258), Synaptophysin (D35E4) XP® Rabbit mAb (Cell Signaling, Cat. #5461), Complex II Subunit Monoclonal Antibody (clone 21A11AE7, Life Technology, #439454), Phospho-p70 S6 Kinase (Thr421/Ser424) (Cell Signaling, #9204), S6K(p70) (Cell Signaling, #2217), Phospho-Akt (Ser473) Antibody (Cell Signaling, Cat. #9271s), Akt (pan) (11E7) Rabbit mAb (Cell Signaling, Cat. #4685s), Ube3a (Abgent, #346954), β-Tubulin Antibody (TUB 2.1, Santa Cruz, Cat. #sc-58886) and β-Actin antibody (Sigma, Cat. #A1978). After incubating with corresponding infrared fluorescent secondary antibodies from Li_COR Biosciences (#926–32213 IRDye® 800CW Donkey anti-Rabbit IgG, and #926–68072 IRDye® 680RD Donkey anti-Mouse IgG), the signal of specific protein was detected and quantified on the Odyssey CLx imager (LI-COR).
3
Hippocampal PSD95 Expression Evaluation
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The rats (n=5 per group) were anesthetized by chloral hydrate, and were perfused transaorticly with normal saline and 4% ice-cold paraformaldehyde. The brain was dissected and paraffin-embedded. The consecutive brain coronal sections (4 μm) were dewaxed, rehydrated and subjected to antigen retrieval in citric acid (Solarbio Life Sciences, Beijing, People’s Republic of China). The sections were blocked with goat serum (Beyotime Biotech) and incubated with anti-PSD95 antibody (Abcam) at 4°C overnight. After being washed, the bound antibodies were detected with horseradish peroxidase-conjugated secondary antibody (LI-COR) and visualized with 3,3′-diaminobenzidine tetrahydrochloride (ZSGB Bio, Beijing, People’s Republic of China), followed by counterstaining with hematoxylin. The cornu ammonis areas CA1, CA2 and CA3, and dentate gyrus regions of the hippocampus were photoimaged under a light microscope (magnification ×200). The integrated optical density in the positively stained areas on the section was statistically analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).36 (link)
4
Quantifying Hippocampal PSD-95 Expression
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One out of every 6 free-floating sections from the hippocampal formation was incubated with a rabbit polyclonal anti-PSD-95 antibody (1:1000, Abcam, Cambridge, UK) and with a CY3-conjugated anti-rabbit IgG secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc. Laboratories, Inc., West Grove, PA, USA). For quantification of PSD-95 immunoreactive puncta, images from the stratum oriens of the hippocampus were acquired using a LEICA TCS SL confocal microscope (63X oil immersion objective, NA 1.32; zoom factor = 8 Leica Microsystems; Wetzlar, Germany). Three to four sections per animal were analyzed and puncta counts were performed on a single plan, 1024 × 1024 pixel images. Counting was carried out using Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).
5
PSD95 Expression in Hippocampal Regions
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The mice were perfusion-fixed with PFA, and their brains were removed. The brains were cut from superior colliculus to optic chiasma, conventionally dehydrated, immersed by wax, embedded and sectioned into 5 μm thick slices. After dewaxing and hydration, the sections were subjected to high-pressure antigen repairing. Subsequently, they were blocked and incubated overnight at 4°C with anti-PSD95 antibody (cat#: ab18258, Abcam, United States). Afterward, the sections were incubated with goat anti-rabbit IgG secondary antibody (cat#: SP-9001, ZSGB-BIO, China) for 1 h, horseradish enzyme-labeled streptavidin for 1 h, followed by DAB staining and hematoxylin counterstaining. Images of hippocampal CA1 and CA3 were observed and collected under a 40 × light microscope (Leica, Germany), and the average optical density was analyzed using the Image J software (National Institutes of Health, United States).
6
Western Blot Analysis of Neuronal and Glial Markers
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Protein extracts were separated by 10 % (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membrane. The membrane was incubated with following primary antibodies for anti-total p38/JNK/ERK MAPK, anti-phospho-p38/JNK/ERK MAPK antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Synaptophysin antibody (1:1000, Abcam), anti-PSD95 antibody (1:1000, Abcam), anti-NeuN antibody (1:1000, Millipore), anti-MAP2 antibody (1:1000, Abcam), anti-GFAP antibody (1:5000, Dako, Glostrup, Denmark), anti-KCa3.1 antibody (1:400, Abcam), and anti-β-actin antibody (1:3000, Sigma). Secondary antibodies were horseradish peroxidase-conjugated antibody (1:3000; Amersham Biosciences) for 1 h at room temperature.
7
Immunohistochemical Analysis of BDNF, SYN, and PSD-95
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As deparaffinization finished, slices were repaired with sodium citrate buffer (0.01 mol/L, pH 6.0). After peroxidase inactivation, phosphate-buffered saline washing and 10% goat serum blocking, sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti- BDNF antibody (1: 800, Abcam, UK), anti-SYN antibody (1: 300, Abcam, UK), and anti- PSD-95 antibody (1: 200, Abcam, UK). Then, the tissues were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1: 300, Zhong Shan Jin Qiao, China) for 1 h at 37°C. 3,3′-diaminobenzidine-4 HCl/H2O2 (DAB, Vector Laboratories, Burlingame, CA, USA) was used for visualization. Hematoxylin was counterstained after BDNF and SYN staining. Images of the motor cortex and the ventral horn region of the lumbar spinal cord were obtained using an optical microscope at 200× and 400× view field by Leica Application Suite (Leica Microsystems, Wetzlar, Germany). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was applied to analyze mean optical density values.
8
Western Blot Analysis of BDNF, SYN, and PSD-95
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Tissues in the motor cortex and the lumbar spinal cord (L2–4 level) of rats were selected. After homogenization and centrifugation, the supernatants were diluted with loading buffer. The protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE) and transferred on to polyvinylidene fluoride membranes (Millipore, USA). Then, the membranes were blocked with 5% milk solubilized in Tris-buffered saline with 0.1% Tween 20 at room temperature for 1 h. After that, the membranes were incubated with rabbit anti-BDNF antibody (1: 1000, Abcam, UK), anti-SYN antibody (1: 1000, Abcam, UK), anti- PSD-95 antibody (1: 1000, Abcam, UK), or glyceraldehyde phosphate dehydrogenase (GAPDH) (1: 1000, Proteintech, USA) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1: 2000, Zhong Shan Jin Qiao, China) for 1 h at room temperature. Reacting bands were visualized though enhanced chemiluminescence method. Image J software (National Institutes of Health, Bethesda, Maryland, USA) was applied for semi-quantitative analysis of the density of the immunoreactive bands.
9
Neuroinflammation and Tight Junction Modulation
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Lipopolysaccharide was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). IL-1β, IL-6, TNFα, MCP-1, and ICAM-1 ELISA kits were purchased from Genetimes Technology Inc. (Shanghai, China). Anti-TH antibody, anti-PSD-95 antibody, anti-COX-2 antibody, anti-Claudin-1 antibody, anti-Occludin antibody, and anti-CX43 antibody, anti-HMGB1 antibody, anti-TLR4 antibody, and Cy3-conjugated secondary antibody were the products of Abcam (Cambridge, United Kingdom). Anti-Iba-1 was the product of Wako (Osaka, Japan).
10
Antibody Characterization and Reagent Details
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Anti-MITOL rabbit polyclonal antibody was described previously14 (link). Anti-APP C-terminal, anti-α-tubulin, and anti-β-actin antibodies were from Sigma. Anti-Aβ (D54D2), anti-synaptophysin, and anti-presenilin 1 (PS1) antibodies were from Cell Signaling. Anti-normal mouse IgG and anti-Tom20 antibodies were from Santa Cruz Biotechnology. The anti-PSD-95 antibody was from Abcam. Anti-Aβ (6E10) was from Covance. Anti-human Aβ E22P (11A1) antibody was from IBL. A11, OC antibodies were from Thermo Fisher Scientific. Anti-Iba1 was from Wako. Bis-ANS was from Cayman Chemical Company. DAPT and Aβ40 were from PEPTIDE INSTITUTE, INC. Aβ40 was dissolved in 0.02% ammonia solution at 300 µM. To obtain seed-free Aβ40 monomers, these Aβ solutions were centrifuged at 200,000 × g for 3 h at 4 °C and the supernatants were collected. Thus, 0.02% ammonia solution was used as a control in the experiments using either Aβ40. ThS was from Sigma. ThT and Rifampicin were from Wako. Rifampicin was used according to a previous report40 (link). Thus, 0.5% low-viscosity carboxyl methylcellulose (purchased from Sigma) was treated as control.
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