Fluoromount g mounting medium

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy, Germany, United Kingdom

Fluoromount-G is a water-soluble, synthetic mounting medium designed for use in fluorescence microscopy. It is formulated to maintain the fluorescence of labeled samples and provide a durable, optically clear mounting solution.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (17 mentions) Triton x 100, by Merck Group (9 mentions) Bovine serum albumin (bsa), by Merck Group (8 mentions) Lsm 880 confocal microscope, by Zeiss (8 mentions) Lsm 710 confocal microscope, by Zeiss (7 mentions) Lsm 780 confocal microscope, by Zeiss (6 mentions) Lsm 800, by Zeiss (5 mentions) Hoechst 33342, by Thermo Fisher Scientific (5 mentions) Zen software, by Zeiss (4 mentions) Lsm 700, by Zeiss (4 mentions) Axio observer z1, by Zeiss (3 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Lsm 710, by Zeiss (3 mentions) Lsm 880 microscope, by Zeiss (3 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (3 mentions) Lsm 900 confocal microscope, by Zeiss (3 mentions) Goat serum, by Thermo Fisher Scientific (3 mentions) Phalloidin 488, by Thermo Fisher Scientific (3 mentions) Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (3 mentions)

120 protocols using fluoromount g mounting medium

Immunofluorescence Staining of Cryosections

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Cryosections were washed with PBS for 10 min and incubated in blocking/permeabilisation solution (5% donkey serum, 1% bovine serum albumin, 0.3% Triton X-100, in PBS) at room temperature for 1 h. This was followed by incubation with the primary antibody overnight at 4 °C. After washes with 0.01% Triton X-100 in PBS, stained sections were subsequently incubated with the secondary antibody at room temperature for 2 h. DAPI was used for staining of the cell nuclei. Sections were mounted in Fluoromount-G Mounting Medium (ThermoFisher Scientific).
For in vivo neutrophil depletion experiment, sections were incubated overnight with the primary antibodies and washed with 0.01% TritonX100 in PBS as described above for cryosections. The sections were subsequently incubated with the fluorophore-conjugated secondary antibodies for 4 h at room temperature, washed, stained with DAPI, post-fixed with 4% PFA and mounted with Fluoromount-G Mounting Medium (ThermoFisher Scientific).
For immunostaining of Ly6g (1A8) and histone H3 (citrulline R2 + R8 + R17; ab5103), the snap-frozen sections were fixed in ice-cold methanol for 10 min, washed in PBS for 10 min, and incubated in 5% bovine serum albumin (for blocking) at room temperature for 1 h, before incubation with the primary antibody overnight, as described above.

Yau A.C., Globisch M.A., Onyeogaziri F.C., Conze L.L., Smith R., Jauhiainen S., Corada M., Orsenigo F., Huang H., Herre M., Olsson A.K., Malinverno M., Sundell V., Rezai Jahromi B., Niemelä M., Laakso A., Garlanda C., Mantovani A., Lampugnani M.G., Dejana E, & Magnusson P.U. (2022). Inflammation and neutrophil extracellular traps in cerebral cavernous malformation. Cellular and Molecular Life Sciences, 79(4), 206.

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Cryosectioning and Immunostaining of Lymph Nodes

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Inguinal lymph nodes were harvested, immersed in Optimum Cutting Temperature (OCT) compound (VWR Chemicals), and immediately frozen on dry ice. 35 µm sections were cut using a cryostat (Leica), mounted on Superfrost Ultra Plus slides (Thermo Scientific Gerhard Menzel), fixed in acetone for 10 min at −20 °C, air-dried for 30 min, and stored at −20 °C.
Prior to staining, non-specific antibody binding was blocked using a solution of 1% v/v normal mouse serum, 2% w/v bovine serum albumin (Sigma), and 0.3% v/v Triton-X100 (Sigma). Primary antibodies and secondary reagent were used as detailed in Supplementary Table 1. After final washing, Fluoromount G mounting medium (eBioscience) was applied, followed by a coverslip.
Slides were visualized using a Zeiss LSM 710 confocal microscope. Images were acquired using Zen software (Zeiss). ImageJ (NIH) was used for image processing. Images were analysed in a manner allowing comparison of both overall GC area and proportion of the B cell area occupied by GCs: GC and B cell areas were identified using GL7 and B220 staining respectively, manually defined, and their areas calculated (44 (link)).

Wang C., Hart M., Chui C., Ajuogu A., Brian I.J., de Cassan S.C., Borrow P., Draper S.J, & Douglas A.D. (2016). Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines. The Journal of Immunology Author Choice, 197(4), 1242-1251.

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Immunohistochemistry and Bright-field Analysis

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5-μm sections were deparaffinized and subjected to heat-induced epitope retrieval using low or high pH retrieval solutions (DAKO), incubated with antibodies and mounted in Fluoromount-G mounting medium supplemented with DAPI (eBioscience). The details of antibodies are provided in Table S2. Alternatively, sections were stained using H&E (Harris modified H&E solution; Sigma-Aldrich) and slides were mounted in Aquatex (Merck). Chromogenic detection was performed as previously described (Ragusa et al., 2014 (link)).
All images were taken using confocal Zeiss Zen v11.0.0.0 (Carl Zeiss) and analyzed using Imaris 8 (v.8.0.2; Bitplane) and Photoshop CC v2015.1.2 (Adobe) software. Bright-field microscope images were taken using a Leica microscope MZ16 with a Leica Microsystems DFC 295 camera and LAS v4.2 software (Leica Microsystems).

González-Loyola A., Bernier-Latmani J., Roci I., Wyss T., Langer J., Durot S., Munoz O., Prat-Luri B., Delorenzi M., Lutolf M.P., Zamboni N., Verdeil G, & Petrova T.V. (2022). c-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs. The Journal of Experimental Medicine, 219(12), e20212418.

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Immunostaining of Germinal Centers

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Mice were sacrificed at day 14 after i.m. immunization. The inguinal dLN were excised and immediately frozen in Optimal Cutting Temperature compound (Thermo Scientific, UK) on dry ice. The sectioning was performed using a Cryostat Leica CM3050S (Leica Biosystems, UK). 10 μm sections were taken and fixed on glass slides. Slides were stored in −80 °C before immunostaining.
Prior to staining the slides containing the dLN, sections were fixed in 100% acetone for 10 min at −20 °C and then air dried for 30 min. Slides were rehydrated in PBS for 10 min and then blocked with 5% PBS/BSA for 45 min. After a wash with PBS, the slides were incubated with AlexaFluor-488 conjugated anti-mouse GL7 (eBioscience, UK) (1:100) and PE conjugated anti-mouse B220 (eBioscience, UK) (1:100) antibodies at RT for 1 h. The slides were then washed 3 times for 10 min each with PBS. After this, FLuoromount G mounting medium (eBioscience, UK) was applied to the slides and coverslips were placed. Slides were then visualized using DMI3000B (Leica Microsystems, UK). Digital images of the slides were taken and edited by ImageJ (NIH, USA).

Li Y., Leneghan D.B., Miura K., Nikolaeva D., Brian I.J., Dicks M.D., Fyfe A.J., Zakutansky S.E., de Cassan S., Long C.A., Draper S.J., Hill A.V., Hill F, & Biswas S. (2016). Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology. Scientific Reports, 6, 18848.

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Immunostaining and Confocal Microscopy

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Sections were cut at 5-μm thickness, deparaffinized, and subjected to heat-induced epitope retrieval using low- or high-pH retrieval solutions (DAKO), incubated with antibodies, and mounted in Fluoromount-G mounting medium supplemented with DAPI (eBioscience).
Images were taken using confocal Zeiss Zen v11.0.0.0 (Carl Zeiss) and analyzed using Imaris 8 (v.8.0.2) (Bitplane) and Photoshop CC v2015.1.2 (Adobe) software. Large image tiles (omental tissue pictures) were acquired using a motorized Zeiss time-lapse Axio Observer.Z1 with Zeiss Axiovision v4.8.2.0 software (Carl Zeiss). Bright-field microscope images were taken using a Leica MZ16 microscope with a Leica Microsystems DFC 295 camera and LAS v4.2 software (Leica Microsystems). The antibodies used for immunostaining are listed in table S7.

González-Loyola A., Bovay E., Kim J., Lozano T.W., Sabine A., Renevey F., Arroz-Madeira S., Rapin A., Wypych T.P., Rota G., Durot S., Velin D., Marsland B., Guarda G., Delorenzi M., Zamboni N., Luther S.A, & Petrova T.V. (2021). FOXC2 controls adult lymphatic endothelial specialization, function, and gut lymphatic barrier preventing multiorgan failure. Science Advances, 7(29), eabf4335.

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Visualizing Germinal Centers in Lymph Nodes

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Inguinal lymph nodes were harvested, immersed in Optimum Cutting Temperature (OCT) compound (VWR Chemicals), and immediately frozen on dry ice. Thirty-five-micrometer sections were cut using a cryostat (Leica), mounted on Superfrost Ultra Plus slides (Thermo Scientific Gerhard Menzel), fixed in acetone for 10 min at −20°C, air-dried for 30 min, and stored at −20°C.
Prior to staining, non-specific Ab binding was blocked using a solution of 1% v/v normal mouse serum, 2% w/v BSA (Sigma-Aldrich), and 0.3% v/v Triton X-100 (Sigma-Aldrich). Primary Abs and secondary reagent were used as detailed in Supplemental Table I. After final washing, Fluoromount G mounting medium (eBioscience) was applied, followed by a coverslip.
Slides were visualized using a Zeiss LSM 710 confocal microscope. Images were acquired using Zen software (Zeiss). ImageJ (National Institutes of Health) was used for image processing. Images were analyzed in a manner allowing comparison of both overall GC area and proportion of the B cell area occupied by GCs: GC and B cell areas were identified using GL7 and B220 staining, respectively, manually defined, and their areas were calculated (44 (link)).

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Tissue Fixation and Cryosectioning Protocol

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Tissues were fixed in 4% PFA for 12 hours, equilibrated in 30% sucrose for 48 hours, and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura). Cryosections were cut at 8-μm thickness and fixed with 4% PFA, washed with PBS, permeabilized with 0.5% Triton X-100, and incubated with blocking solution followed by antibody staining. Sections were mounted in Fluoromount-G mounting medium supplemented with 4′,6-diamidino-2-phenylindole (DAPI; eBioscience).

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Immunofluorescence Staining of Myoblasts

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Myoblasts were grown and differentiated on coverslips for 4 days. For Cav3, Cav1, MF20, GM130 staining, cells are fixed with 4% PFA (v/v) (Sigma-Aldrich) for 10 min at RT, quenched in 50 mM NH₄Cl and then permeabilized with 0.2% BSA (v/v) and 0.05% saponin (v/v) (Sigma-Aldrich) in PBS for 20 min. Cells are incubated sequentially with indicated primary and fluorescence-conjugated secondary antibody in permeabilization buffer for 1 h at RT. For pSTAT3 staining, cells are fixed and permeabilized with cold methanol for 15 min at −20 °C. After washes with PBS 0.2% BSA (v/v), cells are incubated sequentially with indicated primary and fluorescence-conjugated secondary antibody in PBS 0.2% (v/v) for 1 h at RT. In both protocols, coverslips are mounted in Fluoromount-G mounting medium (eBioscience) supplemented with 2 µg mL⁻¹ of DAPI (Sigma-Aldrich). Acquisitions of images are done using a spinning disk microscope (inverted Spinning Disk Confocal Roper/Nikon; Camera: CCD 1392 × 1040 CoolSnap HQ2; objective: ×60 CFI Plan Apo VC). Colocalization between markers was quantified by the Manders’ coefficient using ImageJ software colocalization plugin JACoP^{55 (link)}.

Dewulf M., Köster D.V., Sinha B., Viaris de Lesegno C., Chambon V., Bigot A., Bensalah M., Negroni E., Tardif N., Podkalicka J., Johannes L., Nassoy P., Butler-Browne G., Lamaze C, & Blouin C.M. (2019). Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells. Nature Communications, 10, 1974.

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Immunofluorescence Characterization of Cardiomyocytes

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Undifferentiated iPSCs or EB cultures at different differentiation stages as well as 3-month-old CMs were fixed with 4% paraformaldehyde (Sigma–Aldrich) in PBS at room temperature for 20 min and then blocked with 1% bovine serum albumin (Thermo Fisher Scientific) at 4°C overnight. The primary and respective secondary antibodies used are listed in Supplementary Table 4. Nuclei were counter-stained with 4-6-diamino-2-phenylindole (DAPI, 0.4 μg/mL, Sigma–Aldrich). Samples were mounted with Fluoromount-G mounting medium (eBioscience). Antibodies staining against non-transmembrane proteins were additionally treated with 0.1% Triton X-100 (Sigma–Aldrich) in PBS to permeabilize the cell membrane. Fluorescent images were captured with a fluorescence microscope (Zeiss Observer.Z1 or Axio Imager.M2). For some images, the ApoTome modus (Zeiss) was used.

Li W., Stauske M., Luo X., Wagner S., Vollrath M., Mehnert C.S., Schubert M., Cyganek L., Chen S., Hasheminasab S.M., Wulf G., El-Armouche A., Maier L.S., Hasenfuss G, & Guan K. (2020). Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation. Frontiers in Cell and Developmental Biology, 8, 592893.

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VEGF-NRP1 Interaction Imaging Assay

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HUVECs were grown on 0.2 % gelatin--coated coverslips (Thermo Scientific) until they formed a tight monolayer. After starvation with Endopan 3 without supplement for 5--6 h, they were stimulated with VEGF--A 165 (50 ng/ml), NA--RPARPAR (peptide concentration: 1 μM), anti--NRP1 (3 μg/ml), or otherwise specified ligand. Cells were fixed and permeabilized with ice--cold methanol/acetone (50/50), blocked with PBS/BSA 3%, and stained with primary antibodies at 4 °C overnight. The appropriate secondary antibodies were incubated for 1 h at room temperature. Nuclei were counterstained with Hoechst. The coverslips were mounted in Fluoromount G mounting medium (eBioscience). Stainings were acquired on a motorized inverted Observer.Z1 (Zeiss) or a Zeiss lsm 710 confocal microscope. At least 3 microscopic fields were analyzed per monolayer. With on average 20 cells per microscopic field of view, at least 150 cells were analyzed per experiment.
To inhibit VEGF--A 165 --NRP1 binding, ligand--blocking anti--NRP1 or its control sheep IgG were added to the starved monolayers at 4 °C 30 min before stimulation with VEGF--A 165 . PTK/ZK (100 nM) and nocodazole (20 μM) or the corresponding DMSO controls were added at 37 °C 30 min prior to stimulation.

Roth L., Prahst C., Ruckdeschel T., Savant S., Weström S., Fantin A., Riedel M., Héroult M., Ruhrberg C, & Augustin H.G. (2016). Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation. Science signaling, 9(425).

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