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Mab341

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MAB341 is a laboratory equipment product offered by Merck Group. It is designed for general laboratory use. The core function of MAB341 is to facilitate standard laboratory procedures. Detailed specifications and intended use are not available in this unbiased, factual description.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (3 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions) Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Poly d lysine, by Merck Group (2 mentions) Dnase, by Merck Group (2 mentions) Trypsin, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Urea, by Merck Group (2 mentions) Laminin, by Merck Group (2 mentions) Neurobasal a, by Thermo Fisher Scientific (2 mentions) N ethylmaleimide, by Merck Group (2 mentions) Bovine serum albumin (bsa), by Gemini Bio (2 mentions) Alexafluor 594 streptavidin, by Jackson ImmunoResearch (2 mentions) S6936, by Merck Group (2 mentions)

8 protocols using mab341

1

Immunostaining Protocols for Neuronal Markers

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The following antibodies and dilutions were used: rabbit anti-βIV-spectrin (1:1000, lab-generated16 (link)), rabbit anti-total ankyrin-G (1:1000, lab-generated63 (link)), goat anti-total ankyrin-G (1:1000, lab-generated 16 (link)), rabbit anti-KCNQ2N1 (1:500 64 (link)), rabbit anti-NaV (1:500, Sigma S6936), rabbit anti-neurofascin FNIII (1:500, lab generated 65 (link)), mouse anti-NeuN (1:1000, Sigma MAB377), mouse anti-caspr (1:1000, Neuromab 75-001), guinea pig anti-vGAT (1:1000, Synaptic Systems 131004), mouse anti-GABAA receptor β2-3 (1:1000, Sigma, MAB341), mouse anti-parvalbumin (1:1000, Sigma P3088), rabbit anti-calbindin (1:1000, Swant CB-38a). Antibodies against KCNQ2 were used to label heteromeric KCNQ2/3 channels at the AIS 64 (link). Fluorescently conjugated secondary antibodies Alexa Fluor 488, 568, or 647 (1:250, Life Technologies) and Alexa Fluor 594-Streptavidin (1:1000, Jackson ImmunoResearch 016-580-084). The following reagents were used: FBS, Poly-D-lysine, Laminin, Paraformaldehyde, DNase, Urea, and N-ethylmaleimide were from Sigma-Aldrich. B27 supplement, GlutaMAX, Penicillin-Streptomycin, Neurobasal-A, Hank’s Balanced Salt Solution, Trypsin, Hepes, Lipofectamine 2000 and Prolong Gold Antifade Reagent were from Life Technologies. Bovine serum albumin was from Gemini Bioproducts. Tween 20 was from Calbiochem.
Nelson A., Caballero-Florán R., Díaz J.R., Hull J., Yuan Y., Li J., Chen K., Walder K., Lopez-Santiago L., Bennett V., McInnis M., Isom L., Wang C., Zhang M., Jones K, & Jenkins P. (2018). Ankyrin-G Regulates Forebrain Connectivity and Network Synchronization via Interaction with GABARAP. Molecular psychiatry, 25(11), 2800-2817.
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2

Immunostaining Protocols for Neuronal Markers

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The following antibodies and dilutions were used: rabbit anti-βIV-spectrin (1:1000, lab-generated16 (link)), rabbit anti-total ankyrin-G (1:1000, lab-generated63 (link)), goat anti-total ankyrin-G (1:1000, lab-generated 16 (link)), rabbit anti-KCNQ2N1 (1:500 64 (link)), rabbit anti-NaV (1:500, Sigma S6936), rabbit anti-neurofascin FNIII (1:500, lab generated 65 (link)), mouse anti-NeuN (1:1000, Sigma MAB377), mouse anti-caspr (1:1000, Neuromab 75-001), guinea pig anti-vGAT (1:1000, Synaptic Systems 131004), mouse anti-GABAA receptor β2-3 (1:1000, Sigma, MAB341), mouse anti-parvalbumin (1:1000, Sigma P3088), rabbit anti-calbindin (1:1000, Swant CB-38a). Antibodies against KCNQ2 were used to label heteromeric KCNQ2/3 channels at the AIS 64 (link). Fluorescently conjugated secondary antibodies Alexa Fluor 488, 568, or 647 (1:250, Life Technologies) and Alexa Fluor 594-Streptavidin (1:1000, Jackson ImmunoResearch 016-580-084). The following reagents were used: FBS, Poly-D-lysine, Laminin, Paraformaldehyde, DNase, Urea, and N-ethylmaleimide were from Sigma-Aldrich. B27 supplement, GlutaMAX, Penicillin-Streptomycin, Neurobasal-A, Hank’s Balanced Salt Solution, Trypsin, Hepes, Lipofectamine 2000 and Prolong Gold Antifade Reagent were from Life Technologies. Bovine serum albumin was from Gemini Bioproducts. Tween 20 was from Calbiochem.
Nelson A., Caballero-Florán R., Díaz J.R., Hull J., Yuan Y., Li J., Chen K., Walder K., Lopez-Santiago L., Bennett V., McInnis M., Isom L., Wang C., Zhang M., Jones K, & Jenkins P. (2018). Ankyrin-G Regulates Forebrain Connectivity and Network Synchronization via Interaction with GABARAP. Molecular psychiatry, 25(11), 2800-2817.
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3

Western Blot Analysis of Neuronal Proteins

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Equal amounts of samples measured with BCA assay (Thermo Scientific #23225), were loaded into Bolt 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred to a PVDF membrane (Life Technologies) overnight, blocked with 5% milk (or 5% BSA for surface biotinylation), and incubated with primary antibodies against GABAAR β2/3 (Millipore #MAB341, 1:500), β-PIX (Millipore #07–1450-I, 1:1000), β-actin (Cell Signaling #4970, 1:1000), c-myc (Sigma-Aldrich #4439, 1:1000) and V5 (Cell Signaling #13202, 1:1000) either for 2 hour at room temperature or overnight at 4°C. For western blot experiments after Co-IP experiments that were done using primary neurons, secondary antibodies that were light chain specific were used to avoid imaging heavy chain of IgG (Jackson Immunoresearch Laboratories, #115–605-174; 1:800 and #211–602-171; 1:800). For western blot experiments after surface biotinylation, HRP-linked secondary antibody was used (Cell signaling, #7076, 1:2000). For the rest of the western blot experiments, Alexa Fluor-647 secondary IgG F(ab’)2 fragment antibodies (Cell Signaling, #4414 and #4410; 1:5000) were used. Secondary antibodies were incubated with membrane for 1 hour at room temperature. Blots were imaged using a Chemidoc MP imaging system (Bio Rad) and analyzed using the ImageJ 1.48 gel analyzer tool [53 (link)].
Sahin G.S., Rodriguez-Llamas J.L., Dillon C., Medina I., Appleyard S.M., Gaiarsa J.L, & Wayman G.A. (2021). Leptin increases GABAergic synaptogenesis through the Rho guanine exchange factor β-PIX in developing hippocampal neurons. Science signaling, 14(683), eabe4111.
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4

Quantitative Analysis of Synaptic Proteins

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Primary hippocampal neurons were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min before incubation with primary VGlut1 (Abcam, ab104898, 1:1000), PSD95 (Abcam, ab2723, 1:1000), VGAT (SANTA CRUZ, sc-49574, 1:500) and surface GABAARβ2/3 (Millipore, MAB341, 1:500) antibodies, followed by incubation of Alexa Fluor-conjugated 488 (FITC) and 568 (CY3) secondary antibodies. Z-stacked images were acquired on Olympus FV1000 BX61WI laser scanning confocal microscope. The integrated puncta intensity of these proteins was analyzed using Matlab software. The experiment was repeated at least 3 times from independent cultures.
Liu Z.H., Ding J.J., Yang Q.Q., Song H.Z., Chen X.T., Xu Y., Xiao G.R, & Wang H.L. (2016). Early developmental bisphenol-A exposure sex-independently impairs spatial memory by remodeling hippocampal dendritic architecture and synaptic transmission in rats. Scientific Reports, 6, 32492.
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5

Visualization of GABA Receptor Subunits

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Adult mice were anesthetized using isoflurane, followed by transcardial perfusion of 20 ml ice-cold 4% paraformaldehyde. Brains were removed and postfixed at 4 °C overnight, then cryoprotected in 30% sucrose at 4 °C for 48 hours. Brain sections of 50 µm thickness were cut using a microtome and stored at −20 °C prior to immunostaining. Slices were incubated in rabbit monoclonal anti-HA antibody (1:500; Cell Signaling, #3724) in PBS with 0.3% Triton X and 4% horse serum at 4 °C for 48 h, followed by 2 h incubation in IRDye800-conjugated donkey anti-rabbit IgG secondary antibody (1:1000; Li-COR). Immunolabeled slices were mounted using glycerol/propyl gallate and scanned with a resolution of 42 µm using the Odyssey imaging system (LI-COR). To study the cellular expression of GABAA receptor subunits, the brains were fixed in 4% paraformaldehyde for 30 min, and maintained in 30% sucrose before sectioning on a cryostat at 30 µm before staining. The rabbit anti-α1 antibody (1:100; Abcam, #ab33299), the mouse anti-β2/3 (1:200 ; Millipore, #MAB341) antibody, and the nuclei staining reagent To-pro-3 (1:500) were used 20 (link). The images were acquired under confocal microscopy with objective of 63X and analyzed by ImageJ.
Huang X., Zhou C., Tian M., Kang J.Q., Shen W., Verdier K., Pimenta A, & Macdonald R.L. (2017). OVEREXPRESSING WILD-TYPE γ2 SUBUNITS RESCUED THE SEIZURE PHENOTYPE IN GABRG2+/Q390X DRAVET SYNDROME MICE. Epilepsia, 58(8), 1451-1461.
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6

Immunocytochemistry of Transfected GABA Receptors

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HEK293 cells or COS-7 cells (50,000) were seeded and grown overnight on poly-L-lysine (0.1 mg/ml; P6282-Sigma) coated 13-mm glass coverslips in 24-well plates. Lipofectamine or Effectene was introduced to transfect cells with myc-tagged α1, β3, and γ2 DNA (500 ng) for 24 h. Cells were fixed with paraformaldehyde (4 %; 158127-Sigma-Aldrich)/sucrose (4 %) in PBS (PFA) for 10 min at room temperature. PBS was used to wash cells by rinsing twice and washing for 5 min 3 times. Cells were incubated with glycine (0.3 M) in PBS for 10 min at room temperature to block unreacted PFA, washed with PBS, and incubated with Triton X-100 (0.5%) in PBS to permeabilize the plasma membrane. Cells were incubated with bovine serum albumin (1%; BP9704-100-Fisher-Scientific) in PBS for 30 min at room temperature to block nonspecific binding of antibodies. Primary antibodies (anti-RCAS1 (1:200), anti-CNX (1 μg/ml), anti-α1 (2.6 μg/ml), anti-β2/3 (5 μg/ml; MAB341-Millipore), or anti-myc (2 μg/ml)) were incubated overnight at 4 °C. Cells were washed with PBS, incubated with 1% BSA/PBS to block nonspecific binding, followed by incubation with secondary antibodies and phalloidin for 1 h at room temperature. Cells were washed in PBS, and coverslips were mounted with Prolong antifade reagent (P36930-Invitrogen). Images were acquired with Zeiss LSM 710 confocal microscope.
Yuan B., Hatchett-Walker C., Long P., Xu Z., Stephenson F.A., Haider S, & Jovanovic J.N. (2022). Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits. The Journal of Biological Chemistry, 298(12), 102590.
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7

Internalization of GABAA Receptor Subunits

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Cell-surface receptors were labelled in living cultured neurons or in α1β2-HEK293 cells transiently transfected with the myc-γ2 cDNA, as described previously 19 (link). Briefly, coverslips were incubated with ice-cold Buffer A (mM: 150 NaCl; 3 KCl; 2 MgCl2; 10 HEPES, pH 7.4; 5 Glucose), containing 0.35 M sucrose for 5 minutes, followed by incubation with mouse anti-β2/3 antibody (MAB341-MerckMillipore) for 30 minutes at 4°C in Buffer A containing 0.35 M sucrose, 1 mM EGTA and 1% BSA. Cells were further incubated at 37°C in Buffer A containing 1 mM CaCl2 and 5 µg/ml leupeptin, in the absence or presence of diazepam (1 µM, Tocris), for 1 hour to allow internalization of the labelled β2/3 subunit-containing GABAARs. Cells were fixed with 4% paraformaldehyde/4% sucrose/PBS (PFA/PBS) and processed for immunolabelling with anti-mouse Alexa-555 antibodies overnight at 4°C. Cells were subsequently permeabilized and labelled with anti-mouse Alexa-488 antibodies (Supplementary Table S1) for 1 hour at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope as above.
Nicholson M.W., Sweeney A., Pekle E., Alam S., Ali A.B., Duchen M, & Jovanovic J.N. (2018). Diazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca2+/calcineurin signalling downstream of GABAA receptors. Molecular Psychiatry, 23(9), 1851-1867.
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8

GABA(A) Receptor Internalization Assay

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Cell-surface receptors were labelled in living cultured neurons or in α1β2-HEK293 cells transiently transfected with the myc-γ2 cDNA, as described previously [19 (link)]. Briefly, coverslips were incubated with ice-cold Buffer A (mM: 150 NaCl; 3 KCl; 2 MgCl2; 10 HEPES, pH 7.4; 5 Glucose), containing 0.35 M sucrose for 5 min, followed by incubation with mouse anti-β2/3 antibody (MAB341-MerckMillipore) for 30 min at 4 °C in Buffer A containing 0.35 M sucrose, 1 mM EGTA and 1% BSA. Cells were further incubated at 37 °C in Buffer A containing 1 mM CaCl2 and 5 µg/ml leupeptin, in the absence or presence of diazepam (1 µM, Tocris), for 1 h to allow internalisation of the labelled β2/3 subunit-containing GABAARs. Cells were fixed with 4% paraformaldehyde/4% sucrose/PBS (PFA/PBS) and processed for immunolabelling with anti-mouse Alexa-555 antibodies overnight at 4 °C. Cells were subsequently permeabilized and labelled with anti-mouse Alexa-488 antibodies (Supplementary Table S1) for 1 h at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope as above.
Nicholson M.W., Sweeney A., Pekle E., Alam S., Ali A.B., Duchen M, & Jovanovic J.N. (2018). Diazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca2+/calcineurin signalling downstream of GABAA receptors. Molecular Psychiatry, 23(9), 1851-1867.
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