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Mabe1134

Manufactured by Merck Group
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MABE1134 is a laboratory equipment manufactured by Merck Group. It is designed for general laboratory use. The core function of MABE1134 is to provide a controlled environment for various scientific experiments and procedures.

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Lab products found in correlation

Zoe fluorescent cell imager, by Bio-Rad (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Sp2 spectral confocal microscope, by Leica camera (1 mentions) Alexa fluor 488 conjugated goat polyclonal anti mouse igg antibody a 11029, by Thermo Fisher Scientific (1 mentions) Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions) Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions) Echo revolve microscope, by Olympus (1 mentions) Goat anti mouse igg alexa 488, by Thermo Fisher Scientific (1 mentions) Proteasome inhibitor mg132, by Selleck Chemicals (1 mentions) A1978, by Merck Group (1 mentions) Goat anti mouse igg alexa 568, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Ab15348, by Abcam (1 mentions) Ab9582, by Abcam (1 mentions) Ab76253, by Abcam (1 mentions) Ab7759, by Abcam (1 mentions) Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)

14 protocols using mabe1134

1

SARS-CoV-2 Spike Protein Immunofluorescence Assay

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To infect Calu-3 cells for immunofluorescence assays, the cells were treated with DMSO or 53 μM SRPIN340 for 12 hours before infection. To infect Calu-3 cells, the medium was removed, and the cells were washed with 0.5 ml of PBS. Virus was added at an MOI of 2.5 and the cells were infected at 37°C for 1 hour. After infection, 200 μl of medium and 2x SRPIN340 or DMSO was added to cells and incubated at 37°C for 24 hours. To fix the cells, the culture plate was submerged in 10% NBF for 2 hours. The NBF was removed, and the cells were placed in PBS. For immunostaining, cells were permeabilized with 0.1% Triton X-100 and blocked in PBS, 5% BSA, and 0.1% Tween-20. Cells were stained with either an anti-dsRNA antibody (J2, Sigma MABE1134) at a 1:125 dilution or an anti-SARS-CoV-2 Spike RBD protein antibody (ProSci #9087) at a 1:150 dilution. Alexa Fluor 488– or Alex Fluor 594–conjugated secondary antibodies were used at a 1:1000 dilution. DNA was stained with Hoechst 33342 dye at a 1:10,000 dilution in PBS. Images were captured with a Bio-Rad Zoe fluorescent cell imager.
Yaron T.M., Heaton B.E., Levy T.M., Johnson J.L., Jordan T.X., Cohen B.M., Kerelsky A., Lin T.Y., Liberatore K.M., Bulaon D.K., Van Nest S.J., Koundouros N., Kastenhuber E.R., Mercadante M.N., Shobana-Ganesh K., He L., Schwartz R.E., Chen S., Weinstein H., Elemento O., Piskounova E., Nilsson-Payant B.E., Lee G., Trimarco J.D., Burke K.N., Hamele C.E., Chaparian R.R., Harding A.T., Tata A., Zhu X., Tata P.R., Smith C.M., Possemato A.P., Tkachev S.L., Hornbeck P.V., Beausoleil S.A., Anand S.K., Aguet F., Getz G., Davidson A.D., Heesom K., Kavanagh-Williamson M., Matthews D.A., tenOever B.R., Cantley L.C., Blenis J, & Heaton N.S. (2022). Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication. Science signaling, 15(757), eabm0808.
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2

Immunofluorescence Assay for dsRNA

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Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 15 min at room temperature. Fixed and permeabilized cells were blocked with 2.5% BSA for 15 min and then incubated overnight at 4°C with diluted MabJ2 in PBS/1% BSA (1:500 dilution of the 0.2 mg/mL antibody), which is a specific antibody for dsRNA (clone J2, MABE1134, Merck Sigma, California, USA). Then, the cells were washed and stained with Alexa 546 rabbit anti-mouse IgG secondary antibodies (Thermo Fisher Scientific). Images were captured using a Leica SP2 Confocal Spectral Microscope. The images were processed using Adobe Photoshop.
Huang K.C., Chiang S.F., Chang H.Y., Hong W.Z., Chen J.Y., Lee P.C., Liang J.A., Ke T.W., Peng S.L., Shiau A., Chen T.W., Yang P.C., Chen W.T, & Chao K.S. (2024). Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy. Journal for Immunotherapy of Cancer, 12(5), e008515.
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3

Visualizing SARS-CoV-2 dsRNA Replication

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Cells were fixed in 4% paraformaldehyde for 24 h prior to safe removal from the BSL-3 facility. Fixed cells were permeabilized in 0.2% Triton X-100 in PBS for 15 min at room temperature. After three washes in PBS, cells were blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) in PBS. Cells were immunostained for dsRNA using a mouse monoclonal anti-dsRNA antibody/clone rJ2 (MABE1134; Sigma-Aldrich) overnight at 4°C. After three washes in PBS, primary antibodies were stained for with an Alexa Fluor 488-conjugated goat polyclonal anti-mouse IgG antibody (A-11029; Invitrogen) for 1 h at room temperature. After three washes in PBS, cells were stained for SARS-CoV-2 nucleocapsid with a directly conjugated Alexa Fluor 594 anti-SARS-N antibody (1C7) and for nuclear DNA using 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. After three washes in PBS, cells were imaged by fluorescence microscopy using an EVOS M5000 imaging system (Invitrogen).
Nilsson-Payant B.E., Uhl S., Grimont A., Doane A.S., Cohen P., Patel R.S., Higgins C.A., Acklin J.A., Bram Y., Chandar V., Blanco-Melo D., Panis M., Lim J.K., Elemento O., Schwartz R.E., Rosenberg B.R., Chandwani R, & tenOever B.R. (2021). The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication. Journal of Virology, 95(23), e01257-21.
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4

SARS-CoV-2 Spike Protein Immunofluorescence Assay

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To infect Calu-3 cells for immunofluorescence assays, the cells were treated with DMSO or 53 μM SRPIN340 for 12 hours before infection. To infect Calu-3 cells, the medium was removed, and the cells were washed with 0.5 ml of PBS. Virus was added at an MOI of 2.5 and the cells were infected at 37°C for 1 hour. After infection, 200 μl of medium and 2x SRPIN340 or DMSO was added to cells and incubated at 37°C for 24 hours. To fix the cells, the culture plate was submerged in 10% NBF for 2 hours. The NBF was removed, and the cells were placed in PBS. For immunostaining, cells were permeabilized with 0.1% Triton X-100 and blocked in PBS, 5% BSA, and 0.1% Tween-20. Cells were stained with either an anti-dsRNA antibody (J2, Sigma MABE1134) at a 1:125 dilution or an anti-SARS-CoV-2 Spike RBD protein antibody (ProSci #9087) at a 1:150 dilution. Alexa Fluor 488– or Alex Fluor 594–conjugated secondary antibodies were used at a 1:1000 dilution. DNA was stained with Hoechst 33342 dye at a 1:10,000 dilution in PBS. Images were captured with a Bio-Rad Zoe fluorescent cell imager.
Yaron T.M., Heaton B.E., Levy T.M., Johnson J.L., Jordan T.X., Cohen B.M., Kerelsky A., Lin T.Y., Liberatore K.M., Bulaon D.K., Van Nest S.J., Koundouros N., Kastenhuber E.R., Mercadante M.N., Shobana-Ganesh K., He L., Schwartz R.E., Chen S., Weinstein H., Elemento O., Piskounova E., Nilsson-Payant B.E., Lee G., Trimarco J.D., Burke K.N., Hamele C.E., Chaparian R.R., Harding A.T., Tata A., Zhu X., Tata P.R., Smith C.M., Possemato A.P., Tkachev S.L., Hornbeck P.V., Beausoleil S.A., Anand S.K., Aguet F., Getz G., Davidson A.D., Heesom K., Kavanagh-Williamson M., Matthews D.A., tenOever B.R., Cantley L.C., Blenis J, & Heaton N.S. (2022). Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication. Science signaling, 15(757), eabm0808.
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5

Immunofluorescence Analysis of Viral Proteins

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Cells were seeded on 18mm coverslips a day before infection. After the infection, coverslips were fixed in 4% paraformaldehyde for 20min at RT followed by three PBS(1X) washes. The samples were then blocked with a blocking buffer (3% FCS/PBS (1X)) for 1h at RT while on a shaker. Then the primary antibodies were added to each sample and incubated overnight at 4C. The primary antibodies used included: anti-VP3 (MA5-18206, Thermo Fisher), anti-dsRNA (MABE1134, Sigma-Aldrich) at 1:200 dilution. The following day, the coverslips were washed three times with PBS(1X) for 5min each and the secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594) were added for 1h at RT. Before mounting on microscope slides, cells were additionally washed with PBS(1X). ProLong Glass Antifade Mountant (P36985, Thermo Fisher) with NucBlue was used and the coverslips were allowed to dry overnight before storing at 4C. Images were acquired either using the Echo Revolve microscope or W2 Olympus confocal microscope and analyzed with Fiji software.
Galitska G., Jassey A., Wagner M.A., Pollack N, & Jackson W.T. (2023). Enterovirus D68 capsid formation and stability requires acidic compartments. bioRxiv.
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6

SVV Infection in BHK-21 and PK-15 Cells

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BHK-21 cells and PK-15 cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). The SVV strain CHhb17 and mouse monoclonal antibody against SVV VP1 were preserved in our laboratory (Hou et al., 2019 (link)). Antibodies for β-actin (mouse monoclonal; A1978) and dsRNA antibody (mouse monoclonal; MABE1134) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for hnRNP K (rabbit polyclonal; 11426-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies for histone H3 antibody (rabbit polyclonal; ab183902) and GFP (mouse monoclonal; ab127417) were purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit immunoglobulin (IgG)-horseradish peroxidase (ab205719) and goat anti-mouse IgG-horseradish peroxidase (ab205718) secondary antibodies were purchased from Abcam. Goat anti-rabbit immunoglobulin (IgG)-Alexa-568, goat anti-mouse IgG-Alexa-568, goat anti-mouse IgG-Alexa-488, and goat anti-rabbit IgG-Alexa-488 secondary antibodies were purchased from Molecular Probes (Invitrogen). Proteasome inhibitor MG132 and caspase inhibitor Z-VAD-FMK were purchased from Selleck Chemicals (Shanghai, China).
Song J., Quan R., Wang D, & Liu J. (2022). Seneca Valley Virus 3Cpro Cleaves Heterogeneous Nuclear Ribonucleoprotein K to Facilitate Viral Replication. Frontiers in Microbiology, 13, 945443.
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7

Immunofluorescence Staining of Cultured Cells

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Cultured cells were fixed in 4% PFA (Sigma-Aldrich) in PBS for 10 min and then permeabilized in PBS containing 0.3% Triton X-100 (Sigma-Aldrich). The cultures were then incubated with primary antibodies followed by secondary antibodies (see the dilutions below). 4′,6-diamidino-2-phenylindole (DAPI) (1:1,000) (Thermo Fisher Scientific) was added to visualize cell nuclei. Images were taken using the DMi8 inverted microscope (Leica). The primary antibodies used in the study were as follows: anti-HCG (ab9582, Abcam, 1:200), anti-dsRNA (MABE1134, Merck, 1:200), anti-ACE2 (ab15348, Abcam, 1:200), anti-GATA2 (WH0002624M1, Sigma-Aldrich, 1:100), anti-GATA3 (MA1-028, Invitrogen, 1:100), anti-SDC1 (12922, Cell Signaling Technology, 1:100), anti-DAB2 (ab76253, Abcam, 1:100), anti-MMP2 (40994, Cell Signaling Technology, 1:100), anti-SARS-CoV-2 nucleocapsid (MBS154642, MyBioSource, 1:300) and anti-HLA-G (ab7759, Abcam, 1:50). Secondary antibodies used in the study (all 1:400) were Alexa Fluor 488 goat anti-mouse IgG1 (A21121, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (A31570, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-rabbit IgG (A21428, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG2a (A21137, Thermo Fisher Scientific), Alexa Fluor 647 donkey anti-rabbit IgG (A31573, Thermo Fisher Scientific).
Chen J., Neil J.A., Tan J.P., Rudraraju R., Mohenska M., Sun Y.B., Walters E., Bediaga N.G., Sun G., Zhou Y., Li Y., Drew D., Pymm P., Tham W.H., Rossello F.J., Nie G., Liu X., Subbarao K, & Polo J.M. (2023). A placental model of SARS-CoV-2 infection reveals ACE2-dependent susceptibility and differentiation impairment in syncytiotrophoblasts. Nature Cell Biology, 25(8), 1223-1234.
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8

SARS-CoV-2 Protein and RNA Detection in Tissue Samples

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Paraffin blocks from lungs and brain (ChP, cerebral cortex, globus pallidus, lateral ventricle, medulla oblongata, midbrain, pons, and putamen) were selected to produce a tissue microarray, as adapted from Pires et al. [17] (link). Four-µm sections were deparaffinized and rehydrated, subjected to antigen retrieval using 10 mM citrate buffer (pH 6.0) for 30 min at 98°C, and were then blocked/permeabilized (3% bovine serum albumin/0.3% Triton X-100) for 1 h. Overnight incubation at 4°C was performed with anti-SARS-CoV-2 SP monoclonal antibody (Genetex, Cat GTX632604, 1:500); convalescent serum (CS, 1:1000) or anti-dsRNA (J2) (Merck-Millipore Cat MABE1134, 1:200). The slides were then washed with PBS and incubated with secondary antibody (Goat anti-Mouse Alexa Fluor 488, 1:400; A-11001) for 45 min at 37°C. Nuclei were stained with 0.5 µg/mL 4′-6-diamino-2-phenylindole for 5 minutes, and the slides were mounted with Aqua-Poly-mount (Polysciences, Pennsylvania, USA). Images were acquired with a Leica TCS SP8 confocal microscope using a 63x/oil objective lens.
Gomes I., Karmirian K., Oliveira J.T., Pedrosa C.D., Mendes M.A., Rosman F.C., Chimelli L, & Rehen S. (2021). SARS-CoV-2 infection of the central nervous system in a 14-month-old child: A case report of a complete autopsy. Lancet Regional Health - Americas, 2, 100046.
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9

Antibody-based Visualization of SARS-CoV-2 Targets

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The following antibodies were used: mouse anti‐ACE2 (Santa Cruz, sc‐390851, IF1:200), goat anti‐ACE2 (R&D systems, AF933, WB 1:1000), mouse anti‐dsRNA (Millipore, MABE1134, IF1:100), rabbit anti‐SARS‐CoV‐2 nucleocapsid protein/NP (Sino Biological, 40143‐R040, WB1:1000 IF1:200), rabbit anti‐TMPRSS2 (Abcam, ab92323, WB1:1000), rabbit anti‐cleaved caspase 3 (Cell Signaling, 9661 IF1:30 after pre‐labelling), rabbit anti‐GAPDH (Cell Signaling, 2118, WB1:2000), mouse anti‐ICAM‐1 (R&D systems, BBA3, IF1:200), mouse anti‐ICAM‐1 (Santa Cruz Biotechnology, sc‐8439, WB1:1000), Phalloidin‐Alexa555 (Cytoskeleton, PHDH1‐A, IF1:500), Phalloidin‐Alexa670 (Cytoskeleton, PHDN1‐A, IF1:200). Pre‐labelling of rabbit anti‐cleaved caspase 3 with Alexa Fluor 555 was performed using Zenon Rabbit IgG labelling kit (ThermoFisher, Z‐25305) according to manufacturer protocol.
Schimmel L., Chew K.Y., Stocks C.J., Yordanov T.E., Essebier P., Kulasinghe A., Monkman J., dos Santos Miggiolaro A.F., Cooper C., de Noronha L., Schroder K., Lagendijk A.K., Labzin L.I., Short K.R, & Gordon E.J. (2021). Endothelial cells are not productively infected by SARS‐CoV‐2. Clinical & Translational Immunology, 10(10), e1350.
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10

Immunofluorescence Staining for SARS-CoV-2 Proteins

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Cells were fixed using 4% PFA-PBS for 1h and subsequently washed with PBS. A blocking step was carried out for 1h at room temperature with 10% goat serum/1%BSA in PBS. Nucleocapsid (N) protein detection was performed by primary incubation with human anti-N antibody (Cr3009, 1ug/ml) for 18h, and washed thoroughly in PBS. Where appropriate, N-protein staining was followed by incubation with mouse anti-IRF3 (sc-33641, Santa Cruz) for 1 h. dsRNA was detected by primary incubation with mouse anti-dsRNA (MABE1134, Millipore) for 18h. Primary antibodies were detected by labelling with secondary anti-human AlexaFluor-568 and anti-mouse AlexaFluor 488 conjugates (Jackson Immuno Research) for 1h. All cells were then labelled with either HCS CellMask DeepRed (H32721, Thermo Fisher) or Phalloidin-AlexaFluor 568 (Thermo Fisher) and Hoechst33342 (H3570, Thermo Fisher). Images were acquired using the WiScan® Hermes High-Content Imaging System (IDEA Bio-Medical, Rehovot, Israel) at magnification 10X/0.4NA or 40X/0.75NA. Four channel automated acquisition was carried out sequentially (DAPI/TRITC, GFP/Cy5). For nuclear translocation assay images were acquired at 40X magnification, 35% density/ 30% well area resulting in 102 FOV/well. For dsRNA quantification, images were acquired at 10X magnification, 100% density/ 80% well area resulting in 47 FOV/well.
Thorne L.G., Bouhaddou M., Reuschl A.K., Zuliani-Alvarez L., Polacco B., Pelin A., Batra J., Whelan M.V., Hosmillo M., Fossati A., Ragazzini R., Jungreis I., Ummadi M., Rojc A., Turner J., Bischof M.L., Obernier K., Braberg H., Soucheray M., Richards A., Chen K.H., Harjai B., Memon D., Hiatt J., Rosales R., McGovern B.L., Jahun A., Fabius J.M., White K., Goodfellow I.G., Takeuchi Y., Bonfanti P., Shokat K., Jura N., Verba K., Noursadeghi M., Beltrao P., Kellis M., Swaney D.L., García-Sastre A., Jolly C., Towers G.J, & Krogan N.J. (2021). Evolution of enhanced innate immune evasion by SARS-CoV-2. Nature, 602(7897), 487-495.
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