F4680

Manufactured by Merck Group

Sourced in United States

The F4680 is a precision laboratory instrument designed for analytical tasks. It features advanced technology to deliver accurate and reliable measurements. The core function of the F4680 is to provide precise data and analysis for scientific research and industrial applications.

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F4680 Fluoromount Aqueous mounting medium (2 citations)

26 protocols using f4680

Visualizing Tie2 and FoxO1 in HUVECs

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To assess Tie2 localization and FoxO1 translocation, HUVECs were seeded in 4-well slide chambers (Lab-TekII, 154526, Thermo Fisher) and maintained in Vasculife medium for 24 h. When fully confluent, the HUVECs were treated with 10 μg/ml 4E2, 100 ng/ml ANGPT1, or 100 ng/ml VEGF for 24 h. Thereafter, the cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% BSA in PBS for 40 min, and incubated at 4 °C overnight with primary antibodies: Alexa Fluor 488-conjugated mouse anti-VE-cadherin (16B1, 53-1449-42, Thermo Fisher) or rabbit anti-FoxO1 (C29H4, 2880, Cell Signaling Technology, Danvers). The cells were then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit (A11008, Thermo Fisher) secondary antibody for 1 h, stained with DAPI (564907, BD Biosciences, Franklin Lakes) for 5 min, and embedded in mounting medium (F4680, Sigma-Aldrich). Images were acquired by fluorescence microscopy (EVOS-M5000, Thermo Fisher).

Lee E., Lee E.A., Kong E., Chon H., Llaiqui-Condori M., Park C.H., Park B.Y., Kang N.R., Yoo J.S., Lee H.S., Kim H.S., Park S.H., Choi S.W., Vestweber D., Lee J.H., Kim P., Lee W.S, & Kim I. (2023). An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone. Experimental & Molecular Medicine, 55(2), 470-484.

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Immunohistochemical Profiling of Interneurons

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Parvalbumin, SST and GFP staining were performed on 20–50 µm-thick slices. Briefly, mice were perfused with 0.9% NaCl solution containing Heparin and 4% paraformaldehyde (PFA). Brains were cryo-protected by placing them overnight in 30% sucrose solution and then frozen in Isopentane at a temperature <-50°C. Brains were sliced with a freezing microtome (ThermoFisher HM450). Permeabilization in a blocking solution of PBT with 0.3% Triton and 10% Normal Goat Serum was done at room temperature for 2 hr. Slices were then incubated overnight (4°C) in the same blocking solution containing the primary rabbit anti-PV antibody (1:1000; Thermo Scientific) and mouse anti-SST antibody (1:250; Santa Cruz Biotechnologies). Slices were then rinsed three times in PBS (10 min each) at room temperature and incubated with goat anti-rabbit and a goat anti-mouse antibody (1:500; Jackson IR) coupled to Alexa-488 or 633 for 3.5 hr at room temperature. Slices were then rinsed three times in PBS (10 min each) at room temperature and coverslipped in mounting medium (Fluoromount, Sigma Aldrich F4680). Immunofluorescence was then observed with a slide scanner (Zeiss, Axio Scan.Z1).

Zorrilla de San Martin J., Donato C., Peixoto J., Aguirre A., Choudhary V., De Stasi A.M., Lourenço J., Potier M.C, & Bacci A. (2020). Alterations of specific cortical GABAergic circuits underlie abnormal network activity in a mouse model of Down syndrome. eLife, 9, e58731.

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Immunohistochemical Analysis of Neural Stem Cells

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Paraffin embedded slices were deparaffined and antigen retrieval carried out by incubating the samples in a 10 mM sodium citrate solution at pH 6 + 0.05% Tween20 in distilled water, where they were boiled 3 times, maintained at 95–98 °C for 20 min and finally cooled at room temperature. After blocking in 5% bovine serum albumin buffer, brain samples were incubated overnight with the following primary antibodies: mouse anti-GFAP (1:100, MA5-12023, Thermo Fisher) to label radial-glia/neural stem cells and then assess neurogenic activity close to the ventricular wall; mouse anti-DCX (doublecortin,1:50, sc-271390, Santa Cruz Biotechnology) to identify young neurons/neuroblasts; and mouse anti-Ki67 (1:50, STJ96966, St Johns Labs, UK) to label dividing cells/proliferation. To study the proliferation of neural stem/progenitor cells, we performed a double immunohistochemistry with Ki67 and Sox2 (1:100, Santa Cruz Biotechnology) for cell colocalization. The day after, brain samples were incubated with Alexa Fluor 488 and/or Texas Red secondary antibodies (both 1:300, Thermo Fisher) for 1 h at room temperature in the dark, and, after several washes, slices were mounted (F4680, Sigma). Negative controls received identical treatment except for omission of primary antibodies and showed no specific staining.

Alonso-Alconada D., Gressens P., Golay X, & Robertson N.J. (2023). Therapeutic hypothermia modulates the neurogenic response of the newborn piglet subventricular zone after hypoxia-ischemia. Pediatric Research, 95(1), 112-119.

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Immunofluorescence Staining of Brain Slices

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Free‐floating slices were rinsed in PBS then permeabilized and blocked with PBS‐BT (50 mM Tris–HCl, 150 mM NaCl, 3% bovine serum albumin (BSA), 0.1% Triton X‐100, pH 7.4) blocking solution for 1 h. Afterward, the sections were incubated with primary antibodies (see key resource table) in a 2% normal donkey serum (NDS) and 0.3% Triton X‐100 PBS solution on a shaker overnight at 4°C. The next day, sections were rinsed and incubated with corresponding secondary antibodies directly conjugated with fluorophores (1:500, Cy3, Cy5, and Alexa Fluor 488 conjugate from Jackson ImmunoResearch) for 2 h at room temperature. Finally, slices were rinsed in PBS and mounted (Sigma‐Aldrich, F4680). For DAT and netrin‐1 immunostaining, antigen retrieval was performed in citrate buffer (pH 9) at 80°C.

Jasmin M., Ahn E.H., Voutilainen M.H., Fombonne J., Guix C., Viljakainen T., Kang S.S., Yu L., Saarma M., Mehlen P, & Ye K. (2020). Netrin‐1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson’s disease features. The EMBO Journal, 40(3), e105537.

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Immunohistochemistry for TH and eYFP Proteins

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Detection of TH and eYFP proteins took place according to standard immunohistochemical protocols using primary antibodies [mouse anti-TH (1:1000, Millipore catalog #MAB318, RRID:AB_2201528), chicken anti-GFP (1:1000, Abcam catalog #ab13970, RRID:AB_300798)]. After overnight incubation, primary antibodies were removed and sections were incubated in specific fluorophore-conjugated secondary antibodies (donkey anti-mouse Cy3, Millipore catalog #AP192C, RRID:AB_11214096, donkey anti-chicken A488, Jackson ImmunoResearch catalog #703-545-155, RRID:AB_2340375, both 1:500). Upon rinses, slides were coverslipped using Fluoromount Aqueous mounting medium (Sigma-Aldrich catalog #F4680). For bright-field detection of TH, the peroxidase-based method (ABC kit; Vector Laboratories catalog #PK-4001, RRID:AB_2336810) with DAB chromogen was used. Quantifications were done manually on three mice per group. A stereotaxic atlas (Franklin and Paxinos, 2008 ) was used to outline anatomic borders.

Bimpisidis Z., König N., Stagkourakis S., Zell V., Vlcek B., Dumas S., Giros B., Broberger C., Hnasko T.S, & Wallén-Mackenzie Å. (2019). The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement. eNeuro, 6(3), ENEURO.0066-19.2019.

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Immunofluorescence Staining of G6PD

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Treated cells were seeded on cover glass in 24-well plates. After washing with PBS 3 times, the cells were fixed and permeabilized. Then, 2% BSA was used to block the cells for 1 h at room temperature. Cells were incubated with primary antibody against G6PD (1:100, Proteintech, 25413-1-AP) overnight at 4 °C. After washing with 0.05% Triton X-100 3 times, the cells were incubated with 488-conjugated donkey anti-rabbit IgG (1:1000, Jackson ImmunoResearch Laboratories) for 30 min at room temperature and then with DAPI incubation for 3 min. Finally, the cells were cover-slipped with mounting medium (Sigma, F4680, USA).

Li C., He Z., Yao F., Liao S., Sun K., Sun S., Li Z, & Wang Z. (2023). Role of Escin in breast cancer therapy: potential mechanism for inducing ferroptosis and synergistic antitumor activity with cisplatin. Apoptosis, 28(7-8), 1154-1167.

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Tissue Mounting with Fluoromount

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Tissue sections were washed with PBS for 5 min at RT, allowed to dry, and mounted using fluoromount (Sigma F4680).

Gaspari S., Labouèbe G., Picard A., Berney X., Rodriguez Sanchez‐Archidona A, & Thorens B. (2023). Tmem117 in AVP neurons regulates the counterregulatory response to hypoglycemia. EMBO Reports, 24(8), e57344.

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Immunofluorescence Staining of Endothelial Cells

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HAoECs were seeded onto sterile eight-well chamber slides (Sigma, C7182) at a concentration of 3 × 10⁴ cells/well. Once the cells were confluent (~80%) in 3—4 days, the culture medium was removed, and the monolayers were washed with warm Dulbecco’s phosphate-buffered saline (DPBS). The cells were then fixed with 4% paraformaldehyde (Sigma, 158127) for 20 min at room temperature followed by permeabilization using 0.1% saponin (Sigma, 47036) for 5 min. The cells were then blocked using 1% bovine serum albumin in DPBS for 1 h at room temperature. After blocking, the cells were washed again with DPBS and incubated with the suitable primary antibody dilution overnight at 4 °C in a humidified chamber. The following day, cells were washed with DPBS, and hereafter, the chamber slides were kept in the dark. The cells were then incubated with suitable fluorescence-tagged secondary antibodies (1:500 dilution) at room temperature for 1—2 h. Following incubation, cells were washed with DPBS and stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma, #10236276001) at 1:10,000 dilution for 5 min. Cells were washed with DPBS, and the chamber was removed from the slide. The slide was then mounted with a clean glass coverslip using a fluoromount mounting medium (Sigma, F4680). Immunofluorescence was detected using a Nikon A1R-Ti2 confocal system.

Guha S., Paul C., Alvarez S., Mine Y, & Majumder K. (2020). Dietary γ-Glutamyl Valine Ameliorates TNF-α-Induced Vascular Inflammation via Endothelial Calcium-Sensing Receptors. Journal of agricultural and food chemistry, 68(34), 9139-9149.

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Visualizing Mitochondrial Apoptosis in A549 Cells

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For immunofluorescence microscopic examination, A549 cells were plated on 12-mm poly-l-lysine-coated coverslips and cultured for 24 h and then cells were treated with 3NP (500 μM) for 48 h or transfected with EGFP-BAX expression plasmids using Lipofectamine 2000 (Invitrogen). The culture medium was removed and the prewarmed (37 °C) medium containing LysoTracker was added after 48 h of transfection. Cells were incubated for 1 h under growth conditions and then cells were washed in PBS, fixed with 4% paraformaldehyde in PBS at 4 °C for 10 min and washed again with PBS. The coverslips were mounted on slides with mounting medium (F4680; Sigma) and were examined with a laser scanning confocal microscopy (Nikon, C1S1, Tokyo, Japan).

Guan J.J., Zhang X.D., Sun W., Qi L., Wu J.C, & Qin Z.H. (2015). DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX. Cell Death & Disease, 6(1), e1624-.

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Tissue Staining and Proliferation Assays

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Following deparaffinization, hematoxylin and eosin, and picrosirius red staining (Polysciences, #24901) were performed following manufacturer protocols. Prior to EdU and TUNEL assays, sections were deparaffinized, and incubated with 0.01M Sodium Citrate (pH=6) for 15 min at 95°C for antigen retrieval. A permeabilization step was followed in which the tissue sections were washed with 1% saponin and APBST wash buffer (APBS supplemented with 0.1% Triton-X100). Slides were then incubated in EdU (Invitrogen, #C10337) or TUNEL (Roche #11684795910 or #12156792910) reaction cocktails according to manufacturer guidelines. Slides were then washed 3 times in PBST for 10 min and nuclear counterstain was achieved by incubating slides with Hoechst 33342 (Invitrogen, #H3570) or DAPI (Life technologies, #D1306) at 1:1000 dilution. Slides were then washed in PBST 3 times and mounted with fluorescent mounting media (Sigma, #F-4680).

Tsissios G., Sallese A., Perez-Estrada J.R., Tangeman J.A., Chen W., Smucker B., Ratvasky S.C., Grajales-Esquivel E., Martinez A., Visser K.J., Araus A.J., Wang H., Simon A., Yun M.H, & Rio-Tsonis K.D. (2023). Macrophages modulate fibrosis during newt lens regeneration. bioRxiv.

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