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Mowiol 4 88 reagent

Manufactured by Merck Group
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Mowiol 4–88 is a polyvinyl alcohol (PVA) reagent used in various laboratory applications. It is a water-soluble synthetic polymer with a medium molecular weight and degree of hydrolysis. Mowiol 4–88 can be used as a stabilizing, thickening, or film-forming agent in various laboratory procedures.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Lsm 700, by Zeiss (3 mentions) Axio observer z1, by Zeiss (3 mentions) Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions) Alexa fluor 647 donkey anti mouse, by Jackson ImmunoResearch (2 mentions) Zen blue software, by Zeiss (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Axio imager, by Zeiss (2 mentions) Trap kit, by Merck Group (1 mentions) Alexa 555, by Jackson ImmunoResearch (1 mentions) Alexa 488, by Jackson ImmunoResearch (1 mentions) Axio imager m2 microscope, by Zeiss (1 mentions) 880 confocal microscope, by Zeiss (1 mentions) Ab58803, by Abcam (1 mentions) A1 confocal microscope, by Nikon (1 mentions) Hb 112, by American Type Culture Collection (1 mentions) Eclipse ti inverted microscope, by Nikon (1 mentions) Imaris 8, by Oxford Instruments (1 mentions) Lsm t pmt, by Zeiss (1 mentions)

22 protocols using mowiol 4 88 reagent

1

Immunofluorescence and TRAP staining

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Cells were fixed with 4% paraformaldehyde-PBS for 15min. Following permeabilization and blocking with goat serum, cells were incubated with primary antibodies during 1 hour. Secondary antibodies used were conjugated with Alexa 488 and Alexa 555 from Jackson Immunoresearch (Interchim, Montluçon, France). Samples were mounted using Mowiol 4–88 reagent (Sigma Aldrich) supplemented with DAPI (Life technologies, St Aubin, France) and were analyzed using an upright Axioimager M2 microscope (Carl Zeiss SAS, Le Pecq, France.).
For paraffin-embedded tissues, sections were prepared and immunostained following deparaffinization and hydration. TRAP stainings were performed using the TRAP kit from Sigma Aldrich and according to the manufacturer’s instructions.
Brunner M., Mandier N., Gautier T., Chevalier G., Ribba A.S., Guardiola P., Block M.R, & Bouvard D. (2018). β1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis. PLoS ONE, 13(4), e0196021.
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2

Immunostaining Protocol for MMP9 and Cullin-1

Cited 2 times
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Liver tissues and hepatocytes were treated with 4% PFA/PBS for 30 min at room temperature. Then, samples were treated with 0.25% Triton-X100/PBS for 30 min, followed by the incubation with 4% BSA/PBS for 45 min 22 (link). Primary antibodies against MMP9 (1:1000, #ab58803, Abcam) and Cullin-1 (1:1000, #ab202555, Abcam) and suitable fluorescently-labeled secondary antibodies were sequentially incubated for 2 h, and nuclear staining performed with 5 μg/ml Hoechst 33342 before mounting in Mowiol 4-88 reagent (81381, Sigma). Fluorescence images was captured with a Zeiss 880 confocal microscope using a 100X oil objective, and acquired with same laser output and gain 45 (link).
Li R., Dai Z., Liu X., Wang C., Huang J., Xin T., Tong Y, & Wang Y. (2023). Interaction between dual specificity phosphatase-1 and cullin-1 attenuates alcohol-related liver disease by restoring p62-mediated mitophagy. International Journal of Biological Sciences, 19(6), 1831-1845.
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3

Immunofluorescence Microscopy of Tight Junctions

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Inserts were washed with PBS and incubated in 4% paraformaldehyde (PFA) for 15 min. In a second step, the inserts were incubated in confocal buffer (50 nM ammonium chloride, 0.1% saponin in PBS) for 30 min. Cells were incubated with anti-Zonula occludens 1 (ZO-1, clone 1A12, Thermofisher) and anti-E protein Flavivirus group antibody 4G2 (ATCC, HB-112™) for 2 h followed by the Alexa 633 coupled goat anti-rabbit IgG (Thermofisher), Alexa 488 coupled anti-IgG2a (molecular probes), and anti-β Tubulin directly coupled with Cy3 (clone TUB 2.1, Abcam) in the dark. DAPI (Sigma) was applied to the insert for 5 min and washed with PBS. Each step was performed at room temperature. The membranes were mounted on glass slides in MOWIOL® 4-88 Reagent (Sigma-Aldrich). For confocal microscopy analysis, a Nikon confocal microscope A1 (Nikon) combined with an ECLIPSE Ti inverted microscope (Nikon) and a digital imaging Nikon software (NIS-Elements AR 3.30.02) was used. All images were acquired with the 40X objective and sequential channel acquisition was performed. The images were analyzed with Imaris 8.0.2 software (Bitplane AG, Zurich, Switzerland). To avoid false-positive emissions, different settings were applied including background subtraction, threshold applications, gamma correction, and maxima.
Vielle N.J., García-Nicolás O., Oliveira Esteves B.I., Brügger M., Summerfield A, & Alves M.P. (2019). The Human Upper Respiratory Tract Epithelium Is Susceptible to Flaviviruses. Frontiers in Microbiology, 10, 811.
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4

Immunocytochemical Analysis of Stem Cells

Cited 2 times
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For immunocytochemistry each cell type was cultured
in 24-well plates and fixated in 4% paraformaldehyde.
After rinsing, the samples were permeabilized with
0.1% Triton/phosphate buffered saline (PBS, Sigma,
USA) and unspecific staining sites were blocked with
1% bovine serum albumin (BSA)/PBS. The cells
were incubated overnight with primary antibodies for
Nanog (Abcam, USA) and Klf4 (Cell Signaling, USA).
After rinsing several times with PBS, the cells were
incubated with species-specific secondary antibodies
conjugated to different fluorochromes. Afterwards, the
stained cells were counterstained with DAPI (0.2 μg/
ml 4’, 6 (link)-diamidino-2-phenylindole) (Sigma, USA) for
3 minutes at room temperature and fixed with Mowiol
4-88 reagent (Sigma, USA). As a negative control for
all antibodies, the omission of each primary antibody
in the sample was performed. The labeled cells were
examined with a confocal microscope (Zeiss LSM
700) and images were obtained using a Zeiss LSMTPMT
(1 (link), 2 (link)).
Azizi H., Asgari B, & Skutella T. (2019). Pluripotency Potential of Embryonic Stem Cell-Like Cells Derived from Mouse Testis. Cell Journal (Yakhteh), 21(3), 281-289.
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5

Epidermal and Dermal Whole Mount Staining

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Tail epidermal or dermal whole mounts were prepared as previously described20 (link). Primary antibodies were diluted in PB buffer (0.5% skimmed milk, 0,25% fish gelatin and 1% of Triton X-100 in PBS) and incubated at the following dilutions: anti-Keratin 6A (rabbit, 1:200, BioLegend 905701), anti-Ubiquityl-Histone H2A (Lys119) (clone D27C4) (rabbit, 1:1,000, CST 8240), anti-phospho-S6 Ribosomal Protein (Ser235/236) (clone D57.2.2E) (rabbit, 1:200, CST 4858), anti-Ki67 (rabbit, 1:100, Abcam 16667), anti-E-cadherin (clone 24E10) (rabbit, 1:200, CST 3195), anti-Glut1 (SLC2A1) (EPR3915 clone) (rabbit, 1:200, Abcam ab115730) and Alexa-647 conjugated Phalloidin A (Thermo Fisher). Secondary antibodies together with DAPI (Thermo Fisher, 1 µg ml−1) were incubated 2 h at room temperature. Tissues were mounted using Mowiol 4–88 Reagent (Sigma-Aldrich) mounting solution. MitoTracker staining (MitoTracker Deep Red FM_M22426, Thermo Fisher) was performed in whole-mount epidermis according to datasheet instructions, incubating the samples at 37 °C 10 min in 50 nM MitoTracker solution, before fixation.
Levra Levron C., Watanabe M., Proserpio V., Piacenti G., Lauria A., Kaltenbach S., Tamburrini A., Nohara T., Anselmi F., Duval C., Elettrico L., Donna D., Conti L., Baev D., Natsuga K., Hagai T., Oliviero S, & Donati G. (2023). Tissue memory relies on stem cell priming in distal undamaged areas. Nature Cell Biology, 25(5), 740-753.
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6

Mitochondrial Imaging and Quantification

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Cells were seeded at a density of 2 × 103 cells/mm2 in a 24-well plate. After 24 h, cells were stained with Mitotracker CMX-ROS (Ex/Em: 579/599) which accumulates in active (healthy) mitochondria with intact membrane potentials. Mitotracker CMX-ROS (200 nM) solution was pre-warm before incubating with cells for 40 min at 37°C. Cells then were washed twice in PBS and fixed in cold methanol for 10 min. Blocking was performed in PBS 0.1% Triton X-100 with Fetal Serum Bovine (FBS) at final concentration of 5%. Cells were incubated with primary antibody against Citrate synthase (ab96600, Abcam) over night at 4°C. Bound antibody were detected with 488 Alexa-Fluor-conjugated donkey anti-rabbit IgG antibody (Life Technologies). After two additional washes in PBS 0.1% Triton X-100, cells were incubated with a solution of 300 nM DAPI (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 10 min. The cells were again washed twice with PBS and the microscopy slides were mounted for detection with Mowiol 4-88 Reagent (Millipore, Billerica MA, USA cod. 475,904). Mitochondrial images were acquired with the confocal microscope LSM980 with 63× objective (NA 1.4) using Airyscan detector. Laser wavelength: 405, 488, 561 nm. Quantification of cell area (μm2) and fluorescence intensity was performed using Zen 3.1 software.
Marchese E., Caterino M., Viggiano D., Cevenini A., Tolone S., Docimo L., Di Iorio V., Del Vecchio Blanco F., Fedele R., Simonelli F., Perna A., Nigro V., Capasso G., Ruoppolo M, & Zacchia M. (2022). Metabolomic fingerprinting of renal disease progression in Bardet-Biedl syndrome reveals mitochondrial dysfunction in kidney tubular cells. iScience, 25(11), 105230.
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7

Visualizing Myelination in Cortical Slices

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Cortical slices were washed once with 1x PBS before they were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich, Cat# 16005, USA) in PBS for 1 h at RT. The slices were subsequently rinsed in 1x PBS and blocked in 3% Horse Serum (Gibco, Cat# 26050088), 2% BSA, (PanReac AppliChem, Cat# A1391, Germany), and 0.5% Triton X-100 in 1x PBS for 2 h at RT. Following blocking, the slices were incubated for 48 h with primary antibodies at 4°C. The slices were then washed 3 times for 30 min with blocking solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (1:1,500, Invitrogen, Cat# MP01306, USA) and the appropriate secondary antibodies overnight at 4°C. The slices were washed again 3 times with blocking solution for 30 min and finally mounted on a glass microscope slide using mounting medium containing Mowiol® 4-88 Reagent (Millipore, Cat# 475904, Germany). The primary antibodies used were as follows: against myelin basic protein (rat monoclonal anti-MBP, MCA409S, BioRad, 1:300, USA), Neurofilament-H (chicken anti-NF200, Abcam, Cat# ab4680 1:10,000, UK). Secondary antibodies were all purchased from Life Technologies (Life Technologies, 1:1,000, USA). Images were obtained using confocal laser scanning microscopy (Leica microsystems, TCS SP8, Germany) and processed using Fiji image processing package.1
Kaplanis S.I., Kaffe D., Ktena N., Lygeraki A., Kolliniati O., Savvaki M, & Karagogeos D. (2023). Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis. Frontiers in Cellular Neuroscience, 17, 1201317.
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8

Oil Red O Staining for Liver Lipids

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Oil red O stain was performed to detect liver neutral lipids as previously described (Mehlem et al., 2013 (link)). In brief, tissue sections were processed as described in the previous section. Slides with paired tissues from WT and Pitx2ASE/ASE littermates collected on the same slide were equivalate to room temperature from −80°C when stored, following oil red O and hematoxylin incubation. Slides were rinsed in water before mounted in MOWIOL® 4-88 Reagent (EM475904, EMD Millipore).
Hu S., Mahadevan A., Elysee I.F., Choi J., Souchet N.R., Bae G.H., Taboada A.K., Sanketi B., Duhamel G.E., Sevier C.S., Tao G, & Kurpios N.A. (2021). The asymmetric Pitx2 gene regulates gut muscular-lacteal development and protects against fatty liver disease. Cell reports, 37(8), 110030.
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9

Oil Red O Staining for Liver Lipids

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Oil red O stain was performed to detect liver neutral lipids as previously described (Mehlem et al., 2013 (link)). In brief, tissue sections were processed as described in the previous section. Slides with paired tissues from WT and Pitx2ASE/ASE littermates collected on the same slide were equivalate to room temperature from −80°C when stored, following oil red O and hematoxylin incubation. Slides were rinsed in water before mounted in MOWIOL® 4-88 Reagent (EM475904, EMD Millipore).
Hu S., Mahadevan A., Elysee I.F., Choi J., Souchet N.R., Bae G.H., Taboada A.K., Sanketi B., Duhamel G.E., Sevier C.S., Tao G, & Kurpios N.A. (2021). The asymmetric Pitx2 gene regulates gut muscular-lacteal development and protects against fatty liver disease. Cell reports, 37(8), 110030.
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10

Fluorescence Microscopy of Kidney Sections

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Air-dried kidney sections from unfixed, fresh-frozen tissue were transferred into PBS, followed by blocking in 2% BSA in PBS for 30 min and three 2-min washing steps in PBS for 2 min. Then, the sections were either incubated only with fluorophore-conjugated lectins (2 µg/ml) or in combination with unconjugated or fluorophore-conjugated primary antibodies (1:20,000 rabbit anti-AQP2, 1:2000 rabbit anti-NCC, or 1:20,000 rabbit anti-NKCC2, kindly provided by Jan Loffing, Zurich [43 (link)]; 1:100 mouse anti-AQP2-Alexa488, sc515770 AF588, SCBT; 1:100 mouse anti-LRP2-Alexa546, sc-515772 AF546, SCBT) in PBS for 1 h at RT. After another round of washing, sections with unconjugated primary antibodies were incubated with fluorophore-coupled secondary anti-rabbit antibodies (Cy3 1:250, 711–165-152, or Cy2 1:100, 711–225-152, both Jackson ImmunoResearch) in PBS for another 30 min at RT, followed by three washes in PBS. Finally, the sections were mounted in MOWIOL solution (2.4 g MOWIOL 4–88 reagent (475904; Merck) in 6 g glycerol and 18 ml 0.13 M Tris pH 8.5) for imaging. An overview of lectins used in this study is given in Supplementary Table 1.
Roskosch J., Huynh-Do U, & Rudloff S. (2023). Lectin-mediated, time-efficient, and high-yield sorting of different morphologically intact nephron segments. Pflugers Archiv, 476(3), 379-393.
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