Faramount aqueous mounting media

Manufactured by Agilent Technologies

Faramount Aqueous Mounting Media is a laboratory product designed for use in microscopy applications. It is a water-based medium that can be used to mount and preserve biological samples for observation and analysis.

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Lab products found in correlation

Aec chromogen, by Agilent Technologies (1 mentions) Rabbit ki67, by Abcam (1 mentions) Interleukin 6 (il 6), by Abcam (1 mentions) Cd11b, by BD (1 mentions) High glucose dmem, by Thermo Fisher Scientific (1 mentions) 786 0 cells, by American Type Culture Collection (1 mentions) 3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Indomethacin, by Merck Group (1 mentions) Bx63 microscope, by Olympus (1 mentions) Tcs sp8 mp multiphoton microscope, by Leica camera (1 mentions) A11037, by Thermo Fisher Scientific (1 mentions) Mab4381, by Merck Group (1 mentions) Sc 33759, by Santa Cruz Biotechnology (1 mentions) Ms 295 p, by Thermo Fisher Scientific (1 mentions) Sc 9081, by Santa Cruz Biotechnology (1 mentions) Anti troponin t, by Thermo Fisher Scientific (1 mentions) Lsm 510 confocal microscope, by Zeiss (1 mentions) Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)

9 protocols using faramount aqueous mounting media

Immunohistochemistry of Skin Samples

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A set of full-thickness skin samples were obtained from post-mortem tissue harvesting, fixed overnight in 4% paraformaldehyde, and then dehydrated for paraffin embedding and slide generation; hematoxylin and eosin staining according to standard protocol. For immunohistochemistry, formalin-fixed, paraffin-embedded tissue slides were cleared and rehydrated through a series of ethanol washes. Antigen retrieval (20 min in pressure cooker, microwaved at 100 power) was performed with 10mM citrate. Slides were incubated in hydrogen peroxide (30 min at 4 degrees C) and then blocked with 10% goat serum/0.1% tween for 1 hour at room temperature. Primary antibodies were added and incubated overnight. The following antibodies were used: rabbit Sox9 1:800 (Abcam 185667), rabbit Ki67 1:200 (Abcam 16667), phosphor-EGFR 1:500 (Abcam 40815), IL6 1:500 (Abcam 6672), and CD11b 1:250 (BD Pharmingen 550282). Sections were washed the following day with 0.1% PBST and incubated with rabbit secondary HRP-labeled polymer (Dako) for 1 hour at room temperature, and then quickly washed with 0.1% PBST and PBS. AEC chromogen (Dako) was used for the colometric development reaction. Slides were then briefly counterstained with hemotoxylin, mounted with Faramount Aqueous Mounting Media (Dako) and sealed for subsequent visualization by light microscopy.

Miranda M., Christofk H., Jones D, & Lowry W. (2017). Topical inhibition of the electron transport chain can stimulate the hair cycle. The Journal of investigative dermatology, 138(4), 968-972.

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Adipocyte Differentiation in 786-0 Cells

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786–0 cells (ATCC, Rockville, MD, USA) were maintained at subconfluence in DMEM High Glucose (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% PEST. Cells were seeded in 6-well plates on coverslips and two-days postconfluent cells were treated 48 h with 0.1 uM dexamethasone (Sigma Aldrich, Saint Louis, MO), 10 ug/ml insulin (Sigma Aldrich Saint, Louis, MO), 100 uM indomethacin (Sigma Aldrich, Saint Louis, MO), 500 uM IBMX (Sigma Aldrich, Saint Louis, MO) and 10 uM DAPT (Sigma Aldrich, Saint Louis, MO) or DMSO followed by 12 days of treatment with 10 ug/ml insulin (Sigma Aldrich, Saint Louis, MO) and 10 uM DAPT (Sigma Aldrich, Saint Louis, MO) or DMSO with medium change every second day. Coverslips were washed in PBS and fixed in PFA, stained with Oil Red O and counterstained with Mayer’s hematoxylin according to standard procedures, followed by mounting in Faramount aqueous mounting media (DAKO, Carpenteria, CA). Imaging was performed using Olympus BX63 microscope equipped with UPlanSApo objectives (10x/0.40), (20x/0.75), (40x/0.95) and DP80 camera using the CellSens Dimensions imaging software. Images are representative from triplicate experiments.

Johansson E., Rönö B., Johansson M., Lindgren D., Möller C., Axelson H, & Smith E.M. (2016). Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice. Scientific Reports, 6, 30739.

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Histological Analysis of Bone Tissue

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Femora were fixed in 10% buffered formalin for 48 hours, infiltrated and embedded in methyl methacrylate (Merk Millipore, Vic, AUS). Samples were sectioned (5 μm) as previously described^{9 (link)} and mounted on gelatine coated super frost plus slides. Longitudinal sections were stained with either Toluidine blue or Tartrate Resistant Acid Phosphatase (TRAP) and counterstained with fast green, as previously described^{9 (link)}. Paraffin embedded tibial 5μm sections were rehydrated and mounted in Faramount aqueous mounting media (DAKO). Collagen fibres were visualised using multiphoton and second harmonic generation imaging (Leica TCS SP8MP multiphoton microscope), with an excitation of 880 nm and emission at 440 nm, as previously described^{29 (link)}. Images were processed using Fiji/Image J software^{30 (link)}.

Arthur A., Nguyen T.M., Paton S., Klisuric A., Zannettino A.C, & Gronthos S. (2018). The osteoprogenitor-specific loss of ephrinB1 results in an osteoporotic phenotype affecting the balance between bone formation and resorption. Scientific Reports, 8, 12756.

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Immunofluorescence Characterization of hiPSCs

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hiPSCs were plated onto Matrigel-coated glass coverslips and were fixed using 5% paraformaldehyde for 10 min as previously described³⁶. The coverslips were washed with IF wash buffer (3% PBS containing 0.1% tween-20) for 5 min and blocked using PBS containing 3% BSA for 30 min. Primary antibodies for hiPSC makers (1:100 SOX2, 1:250 Tra-1-81 (mab4381, Millipore), 1:500 Tra-1-60, 1:100 NANOG (SC-33759, Santa Cruz), and 1:100 POU5F1 (OCT4) (SC-9081, Santa Cruz)) were incubated overnight at 4 °C in PBS containing 3% BSA. For immunofluorescent imaging of hiPSC-CMs, anti-Troponin T (1:200 MS-295-P, Thermo-Scientific) and anti-α-sarcomeric actin (1:500, MA1-21597, Thermo-Scientific) were incubated overnight at 4 °C. Coverslips were washed with IF wash buffer 3 time for 5 min and the appropriate secondary was added for 30 min 1:400 (Life technologies, A-11037 or Alexa Fluor 488 goat anti-rabbit IgG H+L, A-11001Alexa Fluor 488 goat anti-mouse IgG H+L). Coverslips were washed for an additional 3 times for 5 min and mounted using Faramount Aqueous Mounting Media (S3025, Dako, Carpinteria, CA). Images were then taken using LSM 510 confocal microscope (Zeiss).

Churko J.M., Lee J., Ameen M., Gu M., Venkatasubramanian M., Diecke S., Sallam K., Im H., Wang G., Gold J.D., Salomonis N., Snyder M.P, & Wu J.C. (2017). Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods. Nature biomedical engineering, 1(10), 826-837.

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Optimized Immunohistochemistry Protocol for Tissue Sections

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Human and murine paraffin-embedded tissue sections used for the immunohistochemistry experiments were sectioned and deparaffinized in xylene followed by isopropanol dilutions. Antigen retrieval was performed with 10 mM sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) following staining with primary antibodies at 4°C overnight. Secondary antibodies were incubated for 60 minutes at room temperature (22°C–24°C). Slides were mounted with Faramount Aqueous Mounting Media (Dako, S3025). See Table 1 for the list of primary and secondary antibodies used.

Lin S.M., Rue R., Mukhitov A.R., Goel A., Basil M.C., Obraztsova K., Babu A., Crnkovic S., Ledwell O.A., Ferguson L.T., Planer J.D., Nottingham A.N., Vanka K.S., Smith C.J., Cantu E I.I.I., Kwapiszewska G., Morrisey E.E., Evans J.F, & Krymskaya V.P. (2023). Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling. The Journal of Clinical Investigation, 134(4), e172116.

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Immunohistochemical Staining of CXCL9

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Tissue slices of 8 μm were fixed with 10% formaldehyde for 5 min, washed with TBS for 3 min and permeabilized with TBS 0.05% Tween-20 for 3 min. Endogenous enzymes were blocked 15 min with Dako dual endogenous enzyme blocker (Dako; S2003). Tissue unspecific binding was avoided by 1 h incubation with 5% bovine serum albumin, 2% powder milk, 1% human IgG in TBS 0.05% Tween-20. Primary antibody, anti-human CXCL9 goat pAb was used at 1 μg ml⁻¹ (R&D; AF392), diluted in REAL antibody diluent (Dako; S2022) and incubated for 1 h. After three washes in TBS 0.05 % Tween-20, the secondary antibody ImmPress anti-goat-polyHRP (Vector; MP-7405) was incubated for 45 min. Slides were washed again three times and then, the substrate AEC + chromogen (Dako; K4005) was incubated for 15 min. Reaction was stopped by immersion in demineralized water. Counterstaining with hematoxilyn (solution according to Mayer; Sigma-Aldrich #51275) was done previous to mounting the slide with Faramount Aqueous Mounting Media (Dako; S3025). Images were acquired using the Mirax Digital Slide Sytem (Zeiss) and analyzed with Image J software.

Gordon-Alonso M., Hirsch T., Wildmann C, & van der Bruggen P. (2017). Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration. Nature Communications, 8, 793.

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Immunohistochemistry and Immunofluorescence of Mouse Tumors

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All methods for IHC and IF of mouse tumors have been described^{33 (link),62 (link)}. Briefly, tumors were excised at days 16–18 after tumor cell inoculation and snap-frozen in Tissue Freezing Medium (OCT; Jung) or fixed with 10% neutral buffered formalin (3 h) and paraffin embedded. Sections (5–20 μm) were prepared and used directly (frozen tissue) or deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval in citrate buffer (0.01 M sodium citrate pH 6; 20 min). For immunostaining, the following rabbit antibodies were used: anti-CD31 (MEC13.3, BD Bioscience), -SOD3 (Cloud Clone), -VEC (LS-B2138, LSBio), -HIF-2α (PAI-32216, Thermo Fisher Scientific), and -3-NT (#06–284, Cell Signaling). Sections were incubated with appropriate fluorescently conjugated secondary antibodies (Alexa 488 or 546, Molecular Probes) or with peroxidase-labeled anti-rabbit IgG (Dako), followed by amino ethyl carbazol (AEC; Enzo) and hematoxylin counterstaining. Sections were mounted with 4,6-diamidino-2-phenylindole (DAPI)-containing Fluoromount-G (SouthernBiotech; fluorescence analyses) or Dako Faramount Aqueous Mounting Media (Dako; conventional IHC) and analyzed with an Olympus FluoView 1000 confocal microscope with a ×60 1.4 oil plan-Apo objective or with a Leica (DM RB) microscope equipped with an Olympus DP70 camera.

Mira E., Carmona-Rodríguez L., Pérez-Villamil B., Casas J., Fernández-Aceñero M.J., Martínez-Rey D., Martín-González P., Heras-Murillo I., Paz-Cabezas M., Tardáguila M., Oury T.D., Martín-Puig S., Lacalle R.A., Fabriás G., Díaz-Rubio E, & Mañes S. (2018). SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2α. Nature Communications, 9, 575.

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Mouse Skin Tissue Staining Protocol

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Slides with OCT sections were prepared by fixing mouse skin sections in 4% formalin for 5 minutes at room temperature. The sections were then washed with PBS for 10 minutes, and then incubated at 37C with staining solution until they reached desired intensity. Staining solutions were prepared with 50mM Tris pH 7.4 (Fisher), 750uM NAD (Sigma), 80uM phenazine methosulfate (Sigma), 600uM nitrotetrazolium blue chloride (Sigma), 10mM MgCl2 (Sigma), and 10mM of lactate (Sigma). The slides were then counterstained with Brazilliant reagent (Anatech 861) and briefly washed with MilliQ water. Slides were mounted with Faramount Aqueous Mounting Media (Dako) and sealed with clear nail polish for subsequent visualization by light microscopy.

Miranda M., Avila I., Esparza J., Shwartz Y., Hsu Y., Berdeaux R, & Lowry W. (2021). Defining a role for G-Protein Coupled Receptor/cAMP/CRE-Binding Protein Signaling in Hair Follicle Stem Cell activation. The Journal of investigative dermatology, 142(1), 53-64.e3.

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Fisetin Modulates Microglia Activation

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BV2 microglial cells (1 × 10⁴ cells/mL) on 3% gelatin-coated coverslips were treated with fisetin (0–5 µM) for 2 h, and then, stimulated with LPS/ATP for 1 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with p65 and p62 antibodies (1:100 in 10% donkey serum) and treated with Alexa Fluor 647-conjugated secondary antibody. The nuclear counterstaining was performed using DAPI (300 nM), and the slides were mounted with Dako Faramount Aqueous Mounting Media. Fluorescence images were captured using a CELENA S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea).

Molagoda I.M., Athapaththu A.M., Choi Y.H., Park C., Jin C.Y., Kang C.H., Lee M.H, & Kim G.Y. (2021). Fisetin Inhibits NLRP3 Inflammasome by Suppressing TLR4/MD2-Mediated Mitochondrial ROS Production. Antioxidants, 10(8), 1215.

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