Whole brain was homogenised in ice-cold buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors (Promega, Madison, WI, USA) and centrifuged at 10,000 g (30 min at 4°C). Supernatant was stored at −20°C. Antibodies were as follows: anti-PUM1 (1:1000, #12322, Cell Signaling Technology, Danvers, MA, USA), anti-PUM2 (1:1000, ab10361, Abcam, Cambridge, UK), anti-SCN1A (1:1000, ASC-001, Alomone Labs, Jerusalem, Israel), anti-SCN2A (1:1000, ASC-002, Alomone Labs), anti-SCN8A (1:1000, ASC-009, Alomone Labs), anti-GLUR2 (1:2000, AB1768-I, Merck, Darmstadt, Germany) and anti-β-actin (1:5000, ab8227, Abcam). Samples (25 µg of protein) were separated by SDS-PAGE, and protein was transferred to a polyvinylidene difluoride membrane (GE Healthcare). After blocking [0.5% bovine serum albumin (BSA) and 0.05% Tween 20 in Tris-buffered saline (TBS-T)], membrane was incubated overnight (4°C) in primary antibody diluted in 0.5% BSA in TBS-T. Membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2500, #7074, Cell Signaling Technology) in 0.3% BSA in TBS-T, and blots were developed with an Enhanced Chemiluminescent Detection Kit (Pierce, Rockford, IL, USA). Protein band density was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Asc 009
Manufactured by Alomone
Sourced in Germany, Palestine, State of, Israel
ASC-009 is a laboratory reagent used in biochemical research. It is a water-soluble compound that can be used in various experimental applications. The core function of ASC-009 is to serve as a tool for researchers in their investigations, without any specific claims about its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (2 mentions)
Asc 002, by Alomone (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Protease inhibitor, by Promega (1 mentions)
Ab10361, by Abcam (1 mentions)
Asc 001, by Alomone (1 mentions)
Criterion gel, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
A11032, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab5320, by Merck Group (1 mentions)
Mab360, by Merck Group (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Ab9610, by Merck Group (1 mentions)
Dfc500, by Leica (1 mentions)
Mabn69, by Merck Group (1 mentions)
Ab104224, by Abcam (1 mentions)
9 protocols using asc 009
1
Quantitative Protein Expression Analysis
2
Purification and Analysis of Whole Brain Membranes
3
Immunohistochemical Analysis of Optic Nerve and Brain
4
Immunostaining of Voltage-Gated Ion Channels
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The mouse monoclonal neuronal class III β-tubulin antibody (TUJ1; subtype IgG2a; Covance, Princeton) was used as a neuronal marker. Nav1.6 subunits were detected using a rabbit polyclonal anti-Nav1.6 antibody (ASC-009; Alomone Labs). Mouse monoclonal antibodies against Navβ4 (subtype IgG1; clone N168/6), Kv1.2 (subtype IgG2b; clone K14/16) and Caspr (subtype IgG1; clone K65/35) were obtained from the UC Davis/NIH NeuroMab Facility. Mouse monoclonal antibodies were detected by the application of isotype-specific secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG2b, Alexa Fluor 555 goat anti-mouse IgG1 and Alexa Fluor 633 goat anti-mouse IgG2a (Life Technologies). Alexa Fluor conjugated goat or donkey anti-rabbit secondary antibodies were used to detect the rabbit anti-Nav1.6 antibody (Life Technologies).
5
Immunofluorescent Labeling of Neural Markers
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Immunofluorescent reactions were performed on free-floating sections following previously published methods 3 (link). Briefly, sections were preincubated with QuickBlock Blocking Buffer (P0260; Beyotime, Shanghai, China) for 1 h. After washing in PBS (3×10 min), the sections were incubated overnight at 4°C with primary antibodies diluted in QuickBlock Primary Antibody Dilution Buffer (P0262; Beyotime). The primary antibodies used in this study were mouse anti-AnkyrinG (1 : 1000, MABN466; Merck Millipore, Shanghai, China), rabbit anti-NeuN (1 : 3000, ab177487; Abcam, Shanghai, China), and rabbit anti-Nav1.6 (1 : 1000, ASC-009; Alomone Labs, Jerusalem, Israel). After several washes in PBS, sections were incubated with the secondary antibodies in QuickBlock Secondary Antibody Dilution Buffer (P0265; Beyotime) for 2 h at room temperature. The secondary antibodies were goat anti-Rabbit IgG H&L (Alexa Fluor 488) (1 : 1000, ab150077; Abcam) and goat anti-Mouse IgG H&L (Alexa Fluor 555) (1 : 1000, ab150114; Abcam). After secondary antibody incubation and several washes in PBS, sections were mounted on clean glass slides with glycerol and sealed with nail polish. Control sections were labeled simultaneously using the same procedure as described above, with the exception that the primary antibody was substituted with QuickBlock Primary Antibody Dilution Buffer.
6
Multimodal Neuronal Analysis in Tissue Sections
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Fixed brain tissues were gradiently dehydrated using 15% and 30% sucrose in PBS, embedded in OCT, and frozen at −80°C before sectioning. A total of 40‐μm thick slides were sectioned using a freezing microtome and then blocked in a blocking buffer consisting of 4% donkey serum and 0.25% Triton X‐100 for 2 h at room temperature. Floating slides were incubated overnight at 4°C in primary antibodies Guinea Pig anti‐NeuN (Millipore, ABN90P, 1:800), Mouse anti‐PD‐1 (Proteintech, 66220‐1‐Ig, 1:500), and Rabbit anti‐Nav1.6 (Alomone Labs, ASC‐009, 1:200) diluted in blocking buffer. Following 3 washes in PBS, sections were then incubated with secondary antibodies Alexa Fluor® 647 anti‐Guinea IgG (Invitrogen, A‐21450, 1:800), Alexa Fluor® 594 anti‐Mouse IgG (Invitrogen, A‐11005, 1:800), and Alexa Fluor® 488 anti‐Rabbit IgG (Invitrogen, A‐11034, 1:800) for 2 h at room temperature. Then, after washing in PBS for 3 times, sections were incubated with DAPI (Sigma, D9542, 1:1000) diluted in PBS for 15 min before being sealed with ProLong@Diamond antifade reagent (Invitrogen, P36965) and glass covers. Images were obtained using a fluorescence microscope (Nikon AIR).
7
Immunostaining of Mouse Brain Sections
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Mice were transcardially perfused, post-fixed and brains cut into 35–40 µm sections. The used antibodies were: rabbit anti-Nav1.6 (1:250, #ASC-009, Alomone labs), mouse anti-CASPR (1:5000, #75-001, Neuromab), goat anti-rabbit Alexa Fluor 568 (1:400, #A-11011, Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 488 (1:400, #A28175, Thermo Fisher Scientific), and goat anti-mouse Alexa 647 (1:400, #ab150115, Abcam).
8
Isolation and Analysis of Membrane Proteins from Mouse Brain
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Membrane proteins were isolated from whole brain as previously described (41 (link)). Briefly, brain tissue from mice at postnatal day 18 (P18) was homogenized and centrifuged at 3500 g for 10 min, and membrane proteins were pelleted from the supernatant at 100 000 g for 30 min. Protein concentration was measured with the BCA assay (Thermo-Fisher, Waltham, MA). Samples containing 50 µg of protein were incubated at 37°C for 20 min in sample loading buffer prior to electrophoresis on 4–15% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA). After transfer, filters were cut and incubated with polyclonal antibody to Nav1.6 (ASC-009, 1:100; Alomone Labs, Jerusalem, Israel), or with monoclonal antibody to α-tubulin (CLT9002, 1:1000, Cedarlane Labs, Burlington, Canada) as a loading control.
9
Neuronal Protein Expression Analysis
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A rabbit polyclonal antibody against APP (1:1000) (A8717, Sigma), rabbit polyclonal anti‐Nav1.6 (1:200) (ASC‐009, Alomone Laboratories), rabbit polyclonal anti‐Nav1.2 (1:200) (ASC‐002, Alomone Laboratories), BACE1 (1:1000) (ab2077, Abcam), Phospho‐NFAT1 (1:500) (AF8011, Affinity Biosciences), NFAT1 (1:500) (DF7189, Affinity Biosciences), β‐actin (1:2000) (ab8227, Abcam), and γ‐tubulin (1:2000) (T6557; Sigma) were obtained from the respective commercial sources. TTX (1 µM, Sigma), EGTA (0.5 mM, sigma), Aβ1‐42 (5 µM, A‐1163–2, rPeptide), and KB‐R7943 (5 µM, MCE) were purchased.
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