Mab377

Manufactured by Thermo Fisher Scientific

Sourced in United States

MAB377 is a monoclonal antibody product offered by Thermo Fisher Scientific. It is designed for research use and its core function is to serve as a detection reagent in various immunoassay applications.

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Lab products found in correlation

17 protocols using mab377

Antibodies for Neuronal Cell Culture and Brain Tissue

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The following antibodies were used for primary culture neurons (supplier, catalog number and working dilution are indicated): anti-Gad67 mouse IgG2a (Millipore MAB5406, 1:500); anti-somatostatin rat (Millipore MAB354, 1:400); anti-HuCD IgG2B (Thermo 16A11, 1:200); anti-NeuN mouse IgG1 (Thermo MAB377, 1:500). The following antibodies were used for brain sections (supplier, catalog number and working dilution are indicated): anti-GFP chicken polyclonal (Abcam ab13970, 1:2000); anti-dsRED rabbit polyclonal (Tanaka LivingColors 632496, 1:1000) (this antibody also detects mCherry); anti-NeuN mouse monoclonal IgG1 (Thermo MAB377, 1:500); anti-Gad67 mouse monoclonal IgG2a (Millipore MAB5406, 1:200); anti-PV mouse monoclonal IgG1 (Swant PV235, 1:2000); anti-somatostatin rat monoclonal IgG2b (Millipore MAB354, 1:400). Anti-cone arrestin (MilliporeSigma AB15282, 1:100) was used to label cones in mouse retina.

Ling J.P., Bygrave A.M., Santiago C.P., Carmen-Orozco R.P., Trinh V.T., Yu M., Li Y., Liu Y., Bowden K.D., Duncan L.H., Han J., Taneja K., Dongmo R., Babola T.A., Parker P., Jiang L., Leavey P.J., Smith J.J., Vistein R., Gimmen M.Y., Dubner B., Helmenstine E., Teodorescu P., Karantanos T., Ghiaur G., Kanold P.O., Bergles D., Langmead B., Sun S., Nielsen K.J., Peachey N., Singh M.S., Dalton W.B., Rajaii F., Huganir R.L, & Blackshaw S. (2022). Cell-specific regulation of gene expression using splicing-dependent frameshifting. Nature Communications, 13, 5773.

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Neuronal Quantification in Mouse Brain

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At t = 12 days post-injection, mice were deeply anaesthetized with Fatal-Plus at a dose of 90 mg kg⁻¹ and transcardially perfused with 20 ml of PBS, followed by 20 ml of 4% paraformaldehyde solution. Brains were quickly extracted and stored in 4% paraformaldehyde solution at 4 °C for 24 h, and were then transferred to 30% sucrose in PBS solution and allowed to equilibrate for 2 days. Brains were then mounted on a cryostat using OCT and sectioned coronally (50 µm). The floating sections were washed in PBS and stained for neurons using anti-NeuN antibody (Sigma-Aldrich MAB377; 1:500) and Alexa 488 secondary antibody (ThermoFisher A11001; 1:1,000). The sections were mounted on slides with PVA-DABCO. Images were acquired using a Leica DMi8 confocal microscope with a 10× and 20× air objective.

Kreitz J., Friedrich M.J., Guru A., Lash B., Saito M., Macrae R.K, & Zhang F. (2023). Programmable protein delivery with a bacterial contractile injection system. Nature, 616(7956), 357-364.

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Immunohistochemical Analysis of Mouse Brain

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At the termination of the experiments, mice were anesthetized with ketamine/xylazine (90 and 10 mg/kg), and perfused with cold 5 mL PBS and 30 mL of 4% paraformaldehyde via an intra-cardial injection. Mouse brains were collected, post-fixed in 4% paraformaldehyde for 2 days at 4°C, and then infiltrated with 30% sucrose for 5 days at 4°C before embedding in OCT medium. Tissue was serially sectioned at a thickness of 30 μm using a Leica cryostat machine. Detection of induced YPF was enhanced by immunohistochemical staining with chicken anti-GFP antibodies (Aves Labs, Tigard, OR, catalog no. GFP-1020). In addition, sections were incubated with antibodies against GFAP (Thermo Fisher Scientific, Waltham, MA, catalog no. PA110019), DCX (Abcam, San Francisco, CA, catalog no. ab18723), or NeuN (Thermo Fisher Scientific, catalog no. MAB377). Following primary antibody incubation at 4°C overnight, sections were incubated with Alexa Fluor secondary antibodies (goat-anti-chicken 488, catalog no. A11039; and goat-anti-rabbit 568, catalog no. A11011).

McGrath E.L., Gao J., Kuo Y.F., Dunn T.J., Ray M.J., Dineley K.T., Cunningham K.A., Kaphalia B.S, & Wu P. (2017). Spatial and Sex-Dependent Responses of Adult Endogenous Neural Stem Cells to Alcohol Consumption. Stem Cell Reports, 9(6), 1916-1930.

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Neuronal Cell and Brain Tissue Immunostaining

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The following antibodies were used for primary culture neurons (supplier, catalog number and working dilution are indicated): anti-Gad67 ms IgG2a (Millipore MAB5406, 1:500); anti-somatostatin rat (Millipore MAB354, 1:400); anti-HuCD IgG2B (Thermo 16A11, 1:200); anti-NeuN mouse IgG1 (Thermo MAB377, 1:500). The following antibodies were used for brain sections (supplier, catalog number and working dilution are indicated): anti-GFP chicken polyclonal (Abcam ab13970, 1:2000); anti-dsRED rabbit polyclonal (Tanaka LivingColors 632496, 1:1000) (this antibody also detects mCherry); anti-NeuN mouse monoclonal IgG1 (Thermo MAB377, 1:500); anti-Gad67 mouse monoclonal IgG2a (Millipore MAB5406, 1:200); anti-PV mouse monoclonal IgG1 (Swant PV235, 1:2000); anti-somatostatin rat monoclonal IgG2b (Millipore MAB354, 1:400).

Blackshaw S., Ling J.P., Bygrave A.M., Santiago C.P., Carmen‐Orozco R.P., Trinh V., Yu M., Li Y., Han J., Taneja K., Liu Y., Dongmo R., Babola T.A., Parker P.H., Jiang L., Leavey P.J., Smith J.J., Vistein R., Gimmen M.Y., Dubner B., Teodorescu P., arantanos T.K., Ghiaur G., Kanold P.O., Bergles D.E., Langmead B., Sun S., Nielsen K.J., Peachy N., Singh M.S., Dalton W.B., Rajaii F., & Huganir R.L. (2022). Cell-specific regulation of gene expression using splicing-dependent frameshifting.

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Neuronal Cell and Brain Tissue Immunostaining

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Ling J.P., Bygrave A.M., Santiago C.P., Carmen‐Orozco R.P., Trinh V., Yu M., Li Y., Han J., Taneja K., Liu Y., Dongmo R., Babola T.A., Parker P.H., Jiang L., Leavey P.J., Smith J.J., Goff L.A., Gimmen M.Y., Dubner B., Helmenstine E., Teodorescu P., Karantanos T., Ghiaur G., Kanold P.O., Bergles D.E., Langmead B., Sun S., Nielsen K.J., Peachey N.S., Singh M.S., Dalton W.B., Rajaii F., Huganir R.L., & Blackshaw S. (2022). Cell-specific regulation of gene expression using splicing-dependent frameshifting.

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Immunohistochemical Analysis of Brain Sections

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Coronal sections of the brain were stained by standard immunohistochemistry procedures as described previously.²⁵ Briefly, the brain sections were incubated with PBS containing 0.1% Triton X‐100 and 5% goat serum (Gibco) for 30 minutes. Then, the sections were incubated overnight at 4°C with the following antibodies as appropriate: anti‐CYP4A (1:200; Abcam, ab3573), anti‐NeuN (1:200; Millipore, MAB377), anti‐GFAP (1:200; Invitrogen) and anti‐Iba1 (1:200; Invitrogen). Nuclei were labelled with DAPI (1:1000, Invitrogen). All sections were imaged using an A1 Si confocal microscope (Nikon) and analysed using ImageJ.
TUNEL staining was used to detect apoptosis and was performed according to the manufacturer's protocols (Roche). Briefly, the sections were treated with 0.3% hydrogen peroxide for 30 minutes and then incubated with proteinase K for 45 minutes. Thereafter, the sections were immersed in TUNEL reaction solution in the dark for 60 minutes. Finally, the sections were labelled using DAPI (1:1000; Invitrogen), and the ratio of TUNEL‐positive to DAPI‐stained cells was calculated to assess the degree of apoptosis.

Cui W., Wu X., Shi Y., Guo W., Luo J., Liu H., Zheng L., Du Y., Wang P., Wang Q., Feng D., Ge S, & Qu Y. (2020). 20‐HETE synthesis inhibition attenuates traumatic brain injury–induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC‐1α pathway: A translational study. Cell Proliferation, 54(2), e12964.

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Immunofluorescence Staining of Mouse Neural Tissue

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Mouse anti-NeuN (Millipore MAB377; RRID: AB_2333092)

Rabbit anti-GFP (Invitrogen A11122; RRID: AB_221569)

Rabbit anti-Dsred (Takara 632496)

Chicken anti-GFP (Aves GFP 1020; RRID: AB_10000240)

Mouse anti-CGRP (Sigma C7113)

Rabbit anti-Iba1 (Abcam ab178846)

647 conjugated IB4 (Thermo Scientific/Life Technology I32450)

Alexa Fluor 488 Goat anti-mouse (Life technologies a11001)

Alexa Fluor 488 Goat anti-rabbit (Life Technologies a11034)

Alexa Fluor 488 Goat anti-chicken (Life Technologies a11039)

Alexa Fluor 546 Goat anti-mouse (Life Technologies a11030)

Alexa Fluor 546 Goat anti-rabbit (Life Technologies a11035)

Bohic M., Upadhyay A., Eisdorfer J.T., Keating J., Simon R.C., Briones B.A., Azadegan C., Nacht H.D., Oputa O., Martinez A.M., Bethell B.N., Gradwell M.A., Romanienko P., Ramer M.S., Stuber G.D, & Abraira V.E. (2023). A new Hoxb8FlpO mouse line for intersectional approaches to dissect developmentally defined adult sensorimotor circuits. Frontiers in Molecular Neuroscience, 16, 1176823.

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Multimarker Immunostaining of Mouse Tissues

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Tissues were fixed in 10% formalin overnight, then cryoprotected in 30% sucrose. Tissues were then embedded in OCT (TissueTek), and floating sections were cryosectioned at 20μm. Tissues were permeabalized and blocked in 5% Normal Goat Serum in PBS with 0.5% TritonX for two hours. Primary and secondary antibodies were diluted in PBS with 3% Normal Goat Serum. Primary antibody incubation was done overnight, and dilutions were as follow: rabbit anti GFP (1:1000, Molecular Probes, A11122), mouse anti NeuN (1:1000, Chemicon, MAB 377), rabbit anti GFAP (1:500, Invitrogen, 18-0063), rabbit anti Iba1 (1:1000, Wako, 019-19741), goat anti ChAT (1:500, Millipore, AB144), rat anti Ctip2/Bcl11B (1:500, Abcam, ab18465, clone [25B6]). Secondary antibodies were used against the appropriate species: AlexaFluor 488, 555, 647 (1:1000, Invitrogen). DAB staining was performed with the Vectastain Elite ABC kit (Vector Laboratories) according to manufacturer’s instructions. Ctip2 staining was performed as in (25 ). We assessed neuromuscular junction denervation status by immunostaining for pre- and post-synaptic markers, rabbit anti-neurofilament-200 (1:160, Sigma-Aldrich, N4142,) and α- bungarotoxin conjugated to Alexa-555 (1:50, Molecular probes, B35451), respectively. For all immunostaining analysis, at least 6 sections were imaged per mouse, with each section at least 160μm apart.

Stoica L., Todeasa S.H., Cabrera G.T., Salameh J.S., ElMallah M.K., Mueller C., Brown RH J.r, & Miguel S.E. (2016). AAV delivered artificial microRNA extends survival and delays paralysis in an Amyotrophic Lateral Sclerosis mouse model. Annals of neurology, 79(4), 687-700.

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Quantifying TRPV1+ Neurons in DRG

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For the therapeutic setting, a minimum of six and a maximum of eight DRGs per segment (lumbar, thoracic and cervical) were dissected and washed with PBS, stored in 4% paraformaldehyde (PFA) for 1 h and cryoprotected in 30% sucrose overnight. Cryosections (8 µm) were stained with primary antibodies against TRPV1 (1:500, Thermo Fisher Scientific, PA1-29421) and NeuN (1:300, Merck Millipore, MAB377), anti-rabbit Alexa 568 (1:1000, Invitrogen) to visualize TRPV1 staining and anti-mouse Alexa 488 (1:1000, Invitrogen) as a secondary antibody for NeuN. DAPI Fluoromount-G (Biozol) was used to visualize cell nuclei. The fluorescence signal of TRPV1 was detected using an inverted fluorescence microscope (BX51; Olympus) equipped with an Olympus DP50 digital camera. Three replicate pictures of each segment/rat were recorded three times (biological n = 4, technical n = 9, ×20 magnification) and analysed by two blinded investigators using the image analysis software ImageJ (National Institutes of Health, Bethesda, USA). TRPV1^high+ cells were counted and reported as the percentage of all neuronal cells in DRG.

Klimas R., Sgodzai M., Motte J., Mohamad N., Renk P., Blusch A., Grüter T., Pedreiturria X., Gobrecht P., Fischer D., Schneider-Gold C., Reinacher-Schick A., Tannapfel A., Yoon M.S., Gold R, & Pitarokoili K. (2021). Dose-dependent immunomodulatory effects of bortezomib in experimental autoimmune neuritis. Brain Communications, 3(4), fcab238.

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Immunoblot Analysis of RNA-Binding Proteins

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Cell extracts were prepared using radioimmune precipitation assay (RIPA) buffer (Sigma) supplemented with a protease inhibitor cocktail (Roche) as described previously^{15 (link)}. For immunoblot analysis, the lithium dodecyl sulfate (LDS)-denatured and reduced protein samples were resolved on a 4–12% polyacrylamide NuPAGE Bis-Tris gel (Invitrogen) and transferred onto a nitrocellulose membrane. Binding of antibodies was detected by the SuperSignal system (Pierce). The primary Abs used in this study were rabbit polyclonal anti-Rbfox3^{15 (link)} (1:3,000), mouse monoclonal anti-Rbfox3 (1:500, Millipore, MAB377), mouse monoclonal anti-myc (1:5,000, Invitrogen, 46–0603), mouse monoclonal anti-Gapdh (1:5,000, Biodesign, H86504M), rabbit polyclonal anti-Drosha (1:1,000, Abcam, ab12286), and goat polyclonal anti-Dgcr8 (1:1,000, Abcam, ab109098). The antibodies have been validated for immunoblot analysis of mouse samples on the manufactures’ websites.

Kim K.K., Yang Y., Zhu J., Adelstein R.S, & Kawamoto S. (2014). Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs. Nature structural & molecular biology, 21(10), 901-910.

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