Mobu 1

Manufactured by Thermo Fisher Scientific

The MoBU-1 is a multipurpose laboratory instrument designed for various applications. It functions as a vortex mixer, capable of agitating samples to facilitate mixing and resuspension.

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Lab products found in correlation

Bromodeoxyuridine (brdu), by Merck Group (2 mentions) Lsm 710, by Zeiss (2 mentions) Clone 6 11b 1, by Merck Group (1 mentions) Alexa fluor 568, by Thermo Fisher Scientific (1 mentions) Asp175, by Cell Signaling Technology (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Lsm 700 system, by Zeiss (1 mentions) Plan apochromat 40x 1.4 oil objective, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Ab13970, by Abcam (1 mentions) Brdu flow kit, by BD (1 mentions) Fortessa, by BD (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Brdu solution, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions) B35130, by Thermo Fisher Scientific (1 mentions) Ab79351, by Abcam (1 mentions) Ab31940, by Abcam (1 mentions) Ab97959, by Abcam (1 mentions)

11 protocols using mobu 1

Cryosectioning and Immunohistochemistry of Regenerating Spinal Cords

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The tails were fixed in 4% paraformaldehyde (PFA) or MEMFA (0.1 M MOPS pH7.4, 2 mM EGTA, 1 mM MgSO₄, 3.7% formaldehyde) and dehydrated in methanol. Rehydrated samples were embedded in 25% fish gelatin / 20% sucrose and cryosectioned at 12 μm thickness. For non‐regenerating spinal cords, sections were taken at least 250 µm anterior to the amputation plane. For the regenerate, only sections posterior to the amputation plane and anterior to the neural ampulla were considered. The following antibodies were used: rabbit anti‐Sox3 (1:500 at 3 dpa or 1:1,000 at 0 and 5 dpa) and anti‐Myt1 (1:750) were a gift from Nancy Papalopulu; mouse anti‐PCNA was used at 1:500 (PC10, Sigma); rabbit anti‐cleaved caspase 3, Asp175 (1:100, 9661, Cell Signalling Technology), rabbit anti‐pH3 (1:100, 06‐570, Sigma), mouse anti‐BrdU (1:300, clone MoBU‐1, B35128, Invitrogen) and anti‐Acetylated Tubulin (1:1,000, clone 6‐11B1, Sigma). Secondary antibodies were goat anti‐rabbit IgG, Alexa Fluor 488 (A‐11008, Invitrogen) and goat anti‐mouse IgG, Alexa Fluor 568 (A‐11004, Invitrogen).
Whole‐mount in situ hybridisation on embryos and tails was performed as previously described (Harland, 1991 (link)). The foxm1 probe was generated from the TGas064p23 clone linearised with ClaI and transcribed using T7 polymerase.

Pelzer D., Phipps L.S., Thuret R., Gallardo‐Dodd C.J., Baker S.M, & Dorey K. (2021). Foxm1 regulates neural progenitor fate during spinal cord regeneration. EMBO Reports, 22(9), e50932.

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Comprehensive Immunostaining of Tissue Samples

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The following primary antibodies were used: chicken anti-GFP (ab13970, 1:1000; Abcam); mouse anti-BrdU (MoBU-1, 1:100; Invitrogen); rabbit anti-K10 (Poly19054, 1:1,000 BioLegend); rabbit anti-RFP (6000-401-379, 1:1000; Rockland); rat anti-Nidogen (sc-33706, 1:1000; Santa Cruz Biotechnology). Tissues were processed for immunostaining as previously described^4,5 and mounted in ProLong Gold (Invitrogen) with or without 4,6-diamidino-2-phenylindole (DAPI; Life Technologies). Confocal images were taken on a Zeiss LSM700 system using a Plan-Apochromat 40X/1.4 oil objective. Image processing was done using Zeiss Zen and ImageJ software.

Sandoval M., Ying Z, & Beronja S. (2021). Interplay of opposing fate choices stalls oncogenic growth in murine skin epithelium. eLife, 10, e54618.

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BrdU Incorporation in Thymocytes

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Adult mice were injected i.p. with 1.5 mg BrdU (Sigma), and tissues were harvested 18 h after injection. Thymocytes were isolated and stained for flow cytometry as outlined above. BrdU incorporation was detected after cell permeabilization using a BrdU flow kit (BD Biosciences) and staining with an Alexa Fluor 647–conjugated anti-BrdU antibody (MoBU-1; Invitrogen).

James K.D., Cosway E.J., Lucas B., White A.J., Parnell S.M., Carvalho-Gaspar M., Tumanov A.V., Anderson G, & Jenkinson W.E. (2018). Endothelial cells act as gatekeepers for LTβR-dependent thymocyte emigration. The Journal of Experimental Medicine, 215(12), 2984-2993.

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BrdU Incorporation and Colony Formation Assay

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Cells were treated with 10 mM Bromodeoxyuridine (BrdU) 2 hours prior to
trypsinization and fixation with cold ethanol. Cell pellets were incubated in
0.08 % (w/v) pepsin (in 0.1 M HCl) for 20 minutes at 37 °C and
centrifuged. Nuclei were incubated in 2 M HCl for 20 minutes at 37 °C
and 0.1 M Sodium Borate added while vortexing. After centrifugation, pellets
were washed in IFA/Tween 20 (10 mM HEPES, 150 mM NaCl, 4 % Fetal Bovine
Serum, 0.1 % Sodium Azide, 0.5 % Tween 20) and incubated in
primary BrdU antibody (MoBu-1, Invitrogen), washed and incubated in secondary
AlexaFluor488 (Invitrogen). Nuclei were resuspended in IFA with 50μg/mL
propidium iodide for flow cytometry on BD Canto Analyser using FlowJo software.
For colony forming assays, 1000 cells were seeded per well of 12-well tissue
culture plates, with media changed every 48 hours. After 8 days, cells were
fixed with 10 % buffered formalin, stained with 0.1 % crystal
violet for 20 minutes, and plates were scanned electronically for colony counts.
Only colonies meeting a minimum size cut-off were counted.

Best S.A., Nwaobasi A.N., Schmults C.D, & Ramsey M.R. (2016). CCAR2 is required for proliferation and tumor maintenance in human Squamous Cell Carcinoma. The Journal of investigative dermatology, 137(2), 506-512.

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Tracking Germinal Center B Cells

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We followed the method described by Gitlin et al. (2015) (link), with minor modifications. Mice immunized with SRBC 8 d prior were intravenously pulsed with 1 mg 5-ethynyl-2′-deoxyuridinae (EdU, Thermo Fisher Scientific Scientific) in 200 µl of PBS, followed 1 h later by an intraperitoneal injection of 2 mg 5-bromo-2′-deoxyuridinae (BrdU, Millipore Sigma) in 200 µl PBS. Mice were sacrificed 40 min after the BrdU pulse and 10⁶ splenocytes were stained for cell surface markers (anti-B220-AF700, IgD-APC, CD38-PE, and CD95-bio/SA-BV605) followed by an overnight cell fixation and permeabilization using the Cytofix/Cytoperm buffer (BD Pharmingen). Samples were then processed using the BD BrdU flow kit components (BD Pharmingen), except we used the anti-BrdU antibody clone MoBU-1 (Invitrogen) and the Click-iT PLUS EdU-Pacific Blue kit (Thermo Fisher Scientific) according to manufacturers’ instructions, except that we used half the amounts of all components of the click reaction. Samples were acquired on a BD Fortessa and analyzed using Flowjo. GCBC were gated as singlets/live/B220⁺/IgD⁻/Cd38⁻/Fas⁺. Early S-phase GCB cells were labeled as EdU-BrdU⁺, mid/late S-phase cells as EdU⁺BrdU⁺, and post-S phase cells as EdU⁺BrdU⁻.

Litzler L.C., Zahn A., Dionne K.L., Sprumont A., Ferreira S.R., Slattery M.R., Methot S.P., Patenaude A.M., Hébert S., Kabir N., Subramani P.G., Jung S., Richard S., Kleinman C.L, & Di Noia J.M. (2023). Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice. The Journal of Experimental Medicine, 220(9), e20220381.

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Quantifying Cell Proliferation in Fgf10 Mutants

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Pregnant dams (E13.5) were injected once with BrdU solution (50 µg/g body weight, Invitrogen 00-0103), harvested one hour later and fixed overnight in 4% PFA. Heads were infiltrated with sucrose/gelatin and 6 µm cryosections prepared as described above. DNA was denatured by incubating the slides in 1N HCl at 48° for 30 minutes, followed by neutralization in PBS. BrdU was detected as described above using a mouse monoclonal antibody, MoBu-1 (Invitrogen B35128) at 1:100 and Alexa Fluor® 594 goat anti-mouse (Invitrogen A11032). For analysis at E11.5, cryosections were prepared and phospho-Histone H3 was detected as described above. To differentiate between sensory and non-sensory duct domains, we co-stained the sections to detect SOX2 also as described above. E11.5 data came from 8 sections in each of 6 controls and 6 Fgf10 null mutants. E13.5 data came from 5 sections in each of 4 controls and 4 Fgf10 null mutants. Student’s t-test (unpaired, two-tailed; Prism 6.0) was used to compare the mean number of BrdU or phospho-Histone H3-positive cells per unit area in control and Fgf10^−/− samples. SOX2-positive and SOX2-negative domains were considered separately.

Urness L.D., Wang X., Shibata S., Ohyama T, & Mansour S.L. (2015). Fgf10 is required for specification of non-sensory regions of the cochlear epithelium. Developmental biology, 400(1), 59-71.

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Splenocyte Proliferation Assay

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Exponentially growing splenocytes were pulsed with 1 mM BrdU (Sigma) for 30 h, irradiated (30 Gy; 2 h of recovery at 37°C), and then fixed with 70% ethanol. Cells were blocked in 3% BSA in PBS for 30 min and then stained with anti-BrdU monoclononal antibody (1:50; Invitrogen, Mobu-1) for 1 h. Before analysis, propidium iodide was added to a final concentration of 50 μg/mL.

Jiang Q., Paramasivam M., Aressy B., Wu J., Bellani M., Tong W., Seidman M.M, & Greenberg R.A. (2015). MERIT40 cooperates with BRCA2 to resolve DNA interstrand cross-links. Genes & Development, 29(18), 1955-1968.

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Dual-Labeling Cell Cycle Progression

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Cells grown in YES were synchronized in G1 phase and released in the presence of 10 µM EdU. After the first S phase, EdU was removed by washing the cells three times with equal volumes of YES. Before the second S phase 50 µM BrdU was added and kept in the medium until the second S phase was completed. After adding the analogue the cells were incubated in the dark until they were fixed. Cell fixation, zymolase- and HCl-treatment and blocking were as described above. EdU detection was then performed as described above. Primary antibody against BrdU (Invitrogen cat # B-35130, MoBU1) was used at a dilution of 1∶20 and the cells were incubated overnight at 4°C on a rotating wheel. The next day, the cells were washed 3 times with PBS, 2% FCS and 0.05% Tween-20. Secondary anti-mouse IgG1:FITC (AbD Serotec cat.# STAR132F) was added at a dilution of 1∶250. After incubation for 2 hours at room temperature, the cells were washed 3 times with PBS, 2% FCS and 0.05% Tween-20. The cells were mounted and viewed as above.

Anda S., Boye E, & Grallert B. (2014). Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast. PLoS ONE, 9(2), e88629.

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Dual Labeling of DNA Synthesis

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Cells were incubated with 20 µM BrdU for 30 min. After various time intervals, cells were incubated with 20 µM EdU for 30 min. Following fixation with 2% formaldehyde for 10 min and permeabilization with 0.5% Triton X-100 for 10 min, cells were treated with 2 N HCl for 30 min. Subsequently, the click reaction was performed for 30 min in TBS containing 4 mM CuSO₄, 4 µM Alexa 488 azide, and 10 mM sodium ascorbate. Cells were then stained with 1 µg/ml mouse anti-BrdU antibodies (MoBU-1; Thermo Fisher Scientific) followed by incubation with goat anti-mouse Alexa 594 antibodies (Thermo Fisher Scientific). Nuclei were stained with 0.5 µg/ml DAPI for 10 min. Images were obtained with a Zeiss LSM 710 laser scanning confocal microscope with a 20× Plan-Apochromat objective. Image analysis was performed on Sum Slices Projection Images generated from confocal stacks.

Kumagai A, & Dunphy W.G. (2017). MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication. Molecular Biology of the Cell, 28(22), 2998-3012.

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Immunohistochemical Profiling of Murine Brain

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After dewaxing paraffin embedded brain slides with a descending alcohol series, antigen retrieval was performed with a citrate buffer (pH 6.0). Next, brain slides were briefly washed with PBS and then treated with 4 NHCl and 0.1 M sodium borate for 10 min each. After blocking with 10% NGS in 0.3% Triton X-100 in PBS (PBS-T), brain slides were incubated at 4 °C over night with the following primary antibodies diluted in blocking solution: mouse anti-BrdU (MoBU-1) (Thermo Fisher Scientific, #B35128, 1:100), mouse anti-Sox2 (abcam, ab79351, 1:50), rabbit anti-Sox2 (abcam, ab97959, 1:100) and rabbit anti-Tbr1 (abcam, ab31940, 1:200). On the next day, brain slides were incubated with the following secondary antibodies diluted 1:500 in blocking buffer for 1 h at room temperature: goat anti-mouse Alexa Fluor 555 (Cell Signaling, CST4409S) and goat anti-rabbit Alexa Fluor 488 (Cell Signaling, CST4412S). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Roth, 1:1000 from a 1 mg/ml stock solution). After washing brain slides three times with PBS, they were mounted with Mowiol (Merck, 475,904-100GM) and stored at 4 °C in darkness.

Middelkamp M., Ruck L., Krisp C., Sumisławski P., Mohammadi B., Dottermusch M., Meister V., Küster L., Schlüter H., Windhorst S, & Neumann J.E. (2021). Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density. Acta Neuropathologica Communications, 9, 185.

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