Mab1596

Manufactured by Merck Group

Sourced in United States

MAB1596 is a monoclonal antibody product developed by Merck Group. It is designed for use in laboratory research applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach without extrapolation.

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Lab products found in correlation

M9942, by Merck Group (2 mentions) Pierce microplate bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Ab14692, by Abcam (1 mentions) Mab5406, by Merck Group (1 mentions) Mab2263, by Merck Group (1 mentions) A5316, by Merck Group (1 mentions) S5768, by Merck Group (1 mentions) Ab133265, by Abcam (1 mentions) Ab32027, by Abcam (1 mentions) Confocal microscope, by Leica (1 mentions) Ab16659, by Abcam (1 mentions) M4403, by Merck Group (1 mentions) Anti mouse igg alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Abn78, by Merck Group (1 mentions) Mab5258, by Merck Group (1 mentions) Ab19194, by Abcam (1 mentions) Ab80579, by Abcam (1 mentions) Ab57632, by Abcam (1 mentions) Ab107216, by Abcam (1 mentions) Ab10099, by Abcam (1 mentions)

12 protocols using mab1596

Hippocampal Synaptic Protein Quantification

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The hippocampus was homogenized as previously described [43 (link)] with some modifications. Briefly, we used 120 μl of TBS buffer (20 mM Tris-HCl, 137 mM NaCl, pH 7.6) containing protease and phosphatase inhibitors and 1% Triton-X100 in a dounce homogenizer. After 30-min incubation on ice, it was centrifuged at 14000g at 4 °C for 30 min. The supernatant was collected. Protein concentrations were determined (Pierce microplate BCA Protein Assay kit, thermofisher.com). Western blot was used as previously described [44 (link)]. The levels of the synaptic proteins PSD-95 (1:3000, MAB1596, Millipore) and synaptophysin (1:1000, Ab14692, Abcam,) were measured and normalized to beta-actin.

Hansson O., Svensson M., Gustavsson A.M., Andersson E., Yang Y., Nägga K., Hållmarker U., James S, & Deierborg T. (2019). Midlife physical activity is associated with lower incidence of vascular dementia but not Alzheimer’s disease. Alzheimer's Research & Therapy, 11, 87.

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Antibodies Used for Protein Detection

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The following primary antibodies were used in the described experiments: mouse anti-HIP1R (612118, BD Biosciences Pharmingen), rabbit anti-HIP1R (AB9882, Milipore), mouse anti-GluN2B and rabbit anti-GFP (homemade mAb), rabbit anti-GluN2A (ab133265, Abcam), mouse anti-GluA1 (MAB2263, Millipore), rabbit anti-GluA1 (ab131507, Abcam), mouse anti-Myc (A2814, Santa Cruz), rabbit anti-Myc (ab32027, Abcam), mouse anti-MAP2 (M9942, Sigma-Aldrich), mouse anti-GAD67 (MAB5406, Millipore), rabbit anti-γ-aminobutyric acid A receptor (GABA_A)-α1 (2583376, Millipore) mouse anti-postsynaptic density (PSD95; MAB1596, Millipore), mouse anti-synaptophysin (S5768, Sigma-Aldrich), and mouse anti-β-actin (A5316, Sigma-Aldrich).
The secondary antibodies used for western blotting were goat anti-mouse IgG-HRP (31460, Pierce) and goat anti-rabbit IgG-HRP (31420, Pierce). The secondary antibodies used for immunostaining were from Life Technologies (Grand Island, NY, USA).

Peng L., Yang Q., Xu X., Du Y., Wu Y., Shi X., Xu J., Zhu L, & Luo J. (2017). Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons. Frontiers in Molecular Neuroscience, 10, 186.

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Immunostaining of Neuronal Synaptic Proteins

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Cultured neurons were fixed with 4% PFA for 10 min at room temperature, permeabilized with 0.1% triton for 10 min, and blocked with 5% BSA in PBS for 30 min. Primary antibodies were diluted with PBS containing 3% BSA and added to fixed cells for 2 h at room temperature. Anti-β-amyloid 1–16 (Biolegend 6E10, catalog #803014; 1:1000), PSD95 (Millipore, catalog #MAB1596; 1:1000), GluA1-Polyclonal (1:300; Kennedy et al., 2010 (link); Hiester et al., 2017 (link)), Gephyrin (Synaptic Systems catalog #147011; 1:1000), and Bassoon (Synaptic Systems catalog #141004; 1:1000). Cells were washed with PBS in between primary and secondary incubations. Samples were incubated with fluorescently conjugated secondary antibodies (1:2000) for 1 h at room temperature.

Actor-Engel H.S., Schwartz S.L., Crosby K.C., Sinnen B.L., Prikhodko O., Ramsay H.J., Bourne J.N., Winborn C.S., Lucas A., Smith K.R., Dell’Acqua M.L, & Kennedy M.J. (2021). Precision Mapping of Amyloid-β Binding Reveals Perisynaptic Localization and Spatially Restricted Plasticity Deficits. eNeuro, 8(6), ENEURO.0416-21.2021.

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Synaptic Marker Colocalization Protocol

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Synaptic marker colocalization was performed as previously described (60 (link)). Briefly, brain sections were stained using antibodies for synaptophysin (ab16659, Abcam) and PSD-95 (MAB1596, Millipore), marking pre- and postsynaptic terminals, respectively. Imaging was performed with a Leica confocal microscope, using a 63× oil objective with a 4.0 digital zoom. Z-stacks of 5 μm thickness from the middle of the tissues were obtained with a 0.2 μm step size. Z-stacks with pre- and postsynaptic puncta were analyzed using the Spots feature of IMARIS. Spots were generated automatically (with manual adjustment for accurate puncta representation) for each channel separately, and total numbers of spots was recorded for each channel. Eventually, spots were analyzed by the Co-localize Spots MATLAB (MathWorks) plugin. Pre- and postsynaptic puncta were defined as colocalized if their centers were within 200 nm.

Gedam M., Comerota M.M., Propson N.E., Chen T., Jin F., Wang M.C, & Zheng H. (2023). Complement C3aR depletion reverses HIF-1α–induced metabolic impairment and enhances microglial response to Aβ pathology. The Journal of Clinical Investigation, 133(12), e167501.

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Synaptogenesis Modulation by Brain Endothelial Cells

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Neurons on 1 DIV were co-cultured with BMEC or treated with B-CM. After 2 days, neurons were fixed and stained with mouse monoclonal antibody MAP2 (1:200; catalog M4403; Sigma-Aldrich) and then incubated with anti-mouse IgG Alexa Fluor 594-conjugated secondary antibody (1:1000; catalog 21203; Invitrogen, Carlsbad, CA, USA).
To test the effects of BMEC co-culture or B-CM on synaptogenesis, neurons on 12 DIV were co-cultured with BMEC or treated with B-CM for 2 days. On 14 DIV, neurons were fixed and stained for presynaptic and postsynaptic markers VGlut-1 (rabbit anti-VGlut-1, 1:200; catalog 48-2400; Invitrogen) and PSD95 (mouse anti-PSD95, 1:200; catalog MAB1596; Millipore). Alexa-conjugated secondary antibodies (anti-rabbit IgG Alexa Fluor 594, 1:1000; catalog A21207 and anti-mouse IgG Alexa Fluor 488, 1:1000; catalog A21202) were used for detection. The fluorescent images were captured using a confocal microscope (SP8, Leica, Wetzlar, Germany).

Wu K.W., Mo J.L., Kou Z.W., Liu Q., Lv L.L., Lei Y, & Sun F.Y. (2017). Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons. Frontiers in Cellular Neuroscience, 11, 290.

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Protein Expression Analysis Protocol

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The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher). The following antibodies were used for immunohistochemistry: NeuN (1:200; ABN78, rabbit, Millipore) and VPS35 (1:300; ab10099, goat, Abcam). The following ELISA kit was used: Hemoglobin Mouse ELISA Kit (ab157715, Abcam). Roche Lumi-Light substrate (12015200001, Sigma-Aldrich) was used for chemiluminescence.

Simoes S., Neufeld J.L., Triana-Baltzer G., Moughadam S., Chen E.I., Kothiya M., Qureshi Y.H., Patel V., Honig L.S., Kolb H, & Small S.A. (2020). Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans. Science translational medicine, 12(571), eaba6334.

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Immunocytochemical Characterization of Cortical Neurons

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At 10 DIV, the cortical cells grown on poly-l-lysine coverslips (three coverslips [13 mm] for each petri dishes [35 mm]) were fixed in 4% PFA/PBS. The coverslips were washed in phosphate-buffer (0.1 M) and permeabilized in 1% Triton X-100 in blocking phosphate-buffer (1% normal donkey serum) for 1 h and probed with the following primary antibodies: rabbit anti-PSD93 (1:200, Thermo Fisher Scientific 34-4700), mouse anti-PSD95 (1:500, Millipore MAB1596), rabbit anti-GluN1 (1:500, Millipore 07-660), rabbit anti-GluN2A (1:200, Millipore 07-632), rabbit anti-GluN2B (1:200, Upstate 06-600) and mouse anti-beta-Tubulin (1:500, Sigma-Aldrich T8660). After washing, the secondary antibodies in normal donkey serum/PBS were incubated at room temperature for 1 h. To visualize the nuclei, the cells were incubated for 30 min with DAPI (4′,6-diamidino-2-phenylindole) in phosphate-buffered solution (1 µg/ml, Sigma-Aldrich).

Cappelli S., Spalloni A., Feiguin F., Visani G., Šušnjar U., Brown A.L., De Bardi M., Borsellino G., Secrier M., Phatnani H., Romano M., Fratta P., Longone P, & Buratti E. (2022). NOS1AP is a novel molecular target and critical factor in TDP-43 pathology. Brain Communications, 4(5), fcac242.

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Immunohistochemical and Western Blot Analysis of Synaptic Proteins

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For immunohistochemistry, guinea pig polyclonal anti-VGLUT1 and anti-VGLUT2 were purchased from Merck Millipore (#AB5905 and #AB2251-I, respectively, Bilerica, MA, USA). anti-VGLUT1 and anti-VGLUT2 were produced against the peptides corresponding to the C-terminus of rat VGLUT1 and VGLUT2. Mouse monoclonal antibody anti-microtubule-associated protein 2 (MAP2) was obtained from Sigma (#M 4403, clone HM-2, St. Louis, MO, USA) and raised against rat brain MAP2.
For the Western blot, mouse monoclonal anti-PSD-95 was obtained from Merck Millipore (#MAB1596, Bilerica, MA, USA). The antibody was raised against a recombinant rat PSD-95 protein. A mouse monoclonal antibody to Glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) from Thermo Fisher Scientific (#AM4300, clone 6C5, Madrid, Spain) was used as a load control.

Martinez-Galan J.R., Garcia-Belando M., Cabanes-Sanchis J.J, & Caminos E. (2022). Pre- and postsynaptic alterations in the visual cortex of the P23H-1 retinal degeneration rat model. Frontiers in Neuroanatomy, 16, 1000085.

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Antibody Characterization for Cell Imaging

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The following antibodies were used in this work, at the stated concentrations: MCT4, RRID: AB_2189333, cat. sc-50329, Santa Cruz BioTechnology (WB 1:500); IBA1, RRID: AB_839504, cat. 019-19741, FUJIFILM Wako (IF 1:1000, lot CAF6806); CD68, RRID: AB_322219, cat. MCA1957, Bio-Rad (IF 1:400, lot 155083); Synapsin1, RRID: AB_2619772, cat. 106011, Synaptic Systems (WB 1:1000, lot 1-28); Homer1, RRID: AB_887730, cat. 160003, Synaptic Systems (WB 1:1000, IF 1:200, lot 3-61); VGAT, RRID: AB_887872, cat. 131011, Synaptic Systems (WB 1:1000, lot 1-91); Gephyrin, RRID: AB_887719, cat 147111, Synaptic Systems (WB 1:1000, lot 1-25); GluA1, RRID: AB_2113602, cat. AB1504, Merck Millipore (WB 1:1000); GluA2, RRID: AB_2533058, cat. 32-0300, Thermo Fisher Scientific (WB 1:250); Synapsin1, RRID: AB_2721082, cat. 106104, Synaptic Systems (IF 1:200, lot 1-4); VGLUT1, RRID: AB_887880, cat. 135311, Synaptic Systems (WB 1:1000, lot 1-7); PSD95, RRID: AB_2092365, cat. MAB1596, Merck Millipore (WB 1:1000, IF 1:200, lot 3845700); LAMP1, RRID: AB_2134500, cat. 1D4B-c, Developmental Studies Hybridoma Bank (IF 1:100, lot 1-6-22). The MCT4 antibody was KO validated in-house, using MCT4 KO microglial samples.

Monsorno K., Ginggen K., Ivanov A., Buckinx A., Lalive A.L., Tchenio A., Benson S., Vendrell M., D’Alessandro A., Beule D., Pellerin L., Mameli M, & Paolicelli R.C. (2023). Loss of microglial MCT4 leads to defective synaptic pruning and anxiety-like behavior in mice. Nature Communications, 14, 5749.

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Western Blotting of Neuronal Proteins

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Western blotting was done as previously reported [21 (link)]. Primary antibodies were against tau (Tau-5, 1:1000, #AHB0042, Invitrogen), Gapdh (1:1000, #MAB374, EMD Millipore), GFP (1:500, #sc-9996, SantaCruz), NR1 (1:1000, #MAB363, EMD Millipore), NR2B (1:1000, #AB1557, EMD Millipore), PSD-95 (1:1000, #MAB1596, EMD Millipore) and drebrin (1:1000, #D3816, EMD Millipore).

van Hummel A., Bi M., Ippati S., van der Hoven J., Volkerling A., Lee W.S., Tan D.C., Bongers A., Ittner A., Ke Y.D, & Ittner L.M. (2016). No Overt Deficits in Aged Tau-Deficient C57Bl/6.Mapttm1(EGFP)Kit GFP Knockin Mice. PLoS ONE, 11(10), e0163236.

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