The 18 μm brain slices were dried at 42 °C, hydrated by PBS, permeabilized and blocked with 5% BSA in PBST(PBS with 0.5% triton) incubated with primary antibody (goat anti-TNFα 1:200 R&D systems AF-410-NA; goat anti-TMEM119 1:200 abcam ab209064; rabbit anti-AMPAR1 1:100 abcam ab183797; rabbit anti AMPAR2 1:600 abcam ab206293, mouse anti-GAD65 1:200 abcam ab26113) at 4 °C overnight. For secondary antibody, delight donkey anti goat 488(earthox E032231 1:200), delight donkey anti rabbit 594(earthox E032421 1:200), delight goat anti mouse 488(earthox E032210 1:400), delight goat anti rabbit 594(earthox E032420 1:400)were used.
Ab183797
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab183797 is a lab equipment product. It is a tool designed for use in scientific research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
S5768, by Merck Group (2 mentions)
Sola15, by Thermo Fisher Scientific (2 mentions)
Dc 17, by Santa Cruz Biotechnology (2 mentions)
Ab6747, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab209064, by Abcam (1 mentions)
Ab26113, by Abcam (1 mentions)
Ab206293, by Abcam (1 mentions)
Ab13552, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
β glycerophosphate disodium salt, by Merck Group (1 mentions)
L alanyl l glutamine, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab34712, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
6 protocols using ab183797
1
Multimarker Immunofluorescence Labeling of Brain Slices
2
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from cultured bEnd.3 cells and brain tissues using protein lysis buffer (P0013C, Beyotime), containing 1% phosphatase inhibitors (Sigma-Aldrich), and 1% protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 12,000 × g (30 min, 4 °C). The total protein concentration was determined using a BCA protein assay kit (Beyotime, P0012) and was adjusted to 3.5 mg/mL. A total of 30 μg of protein was separated by 4–10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with Tris-buffered saline with Tween-20 (TBST) containing 5% milk and analyzed using monoclonal antibodies against Ki67 (1:2,000, SolA15; eBioscience), Cdk5 (1:2,000, DC 17; Santa Cruz), p-Cdk5 (1:2,000, C-7; Santa Cruz), mouse anti-glutamate receptor 1 (1:2000; ab183797, Abcam), mouse anti-synaptophysin (1:2000; S5768, Sigma-Aldrich), mouse anti-PSD95 (1:2000; ab13552, Abcam), total Tau (D1M9X, 1:2000, 46687, Cell Signaling Technology), phospho-Tau Ser199/202 (1:2000, AB9674; Sigma-Aldrich) and rabbit anti-β-actin (1:2,000, 4970; Cell Signaling Technology). Blots were scanned using Image Quant Las4000mini System. ImageJ software (NIH) was used for the mean intensity of bands.
3
Osteogenic Differentiation Assay
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L-alanyl-L-glutamine and insulin-like growth factor-1 (IGF-1) were purchased from Thermo Fisher (Waltham, MA, USA); Lipofectamine 2000 reagent was obtained from Invitrogen (Carlsbad, CA, USA); β-glycerophosphate disodium salt, dexamethasone, and l-ascorbic acid were obtained from Sigma-Aldrich (St Louis, MO, USA); and primary antibodies against GRIA1 (ab183797), GAPDH (ab8245), and HRP secondary antibody (ab6747) were obtained from Abcam (Cambridge, MA, USA).
4
Protein Expression Analysis of hBMSCs
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First, hBMSCs were lysed with RIPA buffer (Beyotime), and then the protein levels in each sample were measured using a BCA kit (Tiangen Biotechnology, Beijing, China). Equivalent amounts of protein were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore) for Western blot assay. The membrane was then sealed for 1 h with 5% skimmed milk and washed with TBST. The PVDF membrane was incubated with the primary antibody against GRIA1 (ab183797; 1:10,000; Abcam, Cambridge, UK), collagen I (ab34710; 1:10,000; Abcam), collagen II (ab34712; 1:10,000;
Abcam), and GAPDH (ab8245; 1:10,000; Abcam) at 4°C overnight. Then, the membrane was incubated at 25°C for 1 h with horseradish peroxidase (HRP) secondary antibody (ab6747; 1:4,000; Abcam). Protein expression was visualized using an ECL kit (Beyotime) as described previously [23 (link)], and band intensities were quantified using Image J software (NIH, Bethesda, MD, USA).
Abcam), and GAPDH (ab8245; 1:10,000; Abcam) at 4°C overnight. Then, the membrane was incubated at 25°C for 1 h with horseradish peroxidase (HRP) secondary antibody (ab6747; 1:4,000; Abcam). Protein expression was visualized using an ECL kit (Beyotime) as described previously [23 (link)], and band intensities were quantified using Image J software (NIH, Bethesda, MD, USA).
5
Western Blot Analysis of Hippocampal Proteins
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Protein samples harvested from the hippocampus were lysed by cold lysis buffer (RIPA: PMSF = 100:1) on ice. The concentration of protein was quantified by BCA Protein Assay Kit (Beyotime Biological Co., Ltd.). Twenty micrograms protein samples combined with loading buffer were subjected to SDS-PAGE electrophoresis, transferred onto membranes, and blocked by 5% milk in TBST. Subsequently, the membranes were incubated overnight with primary antibodies, including Netrin-1 (1:500, ab126729, Abcam), DCC (1:1,000, ab273570, Abcam), Neo-1 (1:1,000, 20246-1-AP, Proteintech), GluA1 (1:1,000, ab183797, Abcam), NR1 (1:1,000, ab109182, Abcam), NR2A (1:1,000, ab124913, Abcam), NR2B (1:1,000, ab65783, Abcam), PSD95 (1:1,000, ab36233, Abcam), or Tubulin (1:1,000, ARG62347, Arigo Biolaboratories Co.). On the following day, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG antibody (1:5,000, ARG65350/65351, Arigo Biolaboratories Co.) and visualized using an ECL Chemiluminescent Kit (Sigma, United States). Gel image analyses were performed using the ImageJ software.
6
Immunofluorescence Profiling of Neural Markers
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The following primary antibodies were used: mouse anti-Aβ1-42 (1:1,000; A5213, Sigma-Aldrich), rat anti-CD68 (1:400; MCA1957, Bio-Rad), rabbit anti-Iba-1 (1:1,000; Wako Chemical), mouse anti-glutamate receptor 1 (1:400; ab183797, Abcam), mouse anti-synaptophysin (1:200; S5768, Sigma-Aldrich), rat anti-mouse Ly6G (1:200; BP0075, Bioxcell), mouse anti-myeloperoxidase (MPO, 1:1,000; AF3667-SP, R&D), mouse anti neutrophil elastase (NE, 1:1 000; MAB4517-SP, R&D), rat anti-Ki67 (1:500; SolA15, eBioscience), rabbit anti-Claudin5 polyclonal antibody (1:400; YT0953, Immunoway) mouse anti-pCdk5 (1:400; C-7, Santa Cruz) and mouse anti-Cdk5 (1:400; DC 17, Santa Cruz). The following secondary antibodies were used: Alexa Fluor 647 donkey anti-mouse, Alexa Fluor 594 donkey anti-rat, Alexa Fluor 488 donkey anti-goat, Alexa Fluor 555 goat anti-rabbit, and Alexa Fluor 488 goat anti-rabbit (1:400, Invitrogen). PE-conjugated anti-CD31 (1:100; 553373, B&D), FITC-conjugated anti-CXCL1 (1:100; IC4532G, B&D) and DyLight 649-labeled lectin (1:200; L32472, InvitrogenTM) antibodies were used in some immunofluorescence experiments. The detailed immunofluorescence protocols have been described previously [30 (link)].
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