Abc99

Manufactured by Merck Group

The ABC99 is a compact and versatile laboratory equipment designed for a variety of applications. It features precise temperature control and advanced data monitoring capabilities. The core function of the ABC99 is to provide a reliable and consistent environment for conducting scientific experiments and analysis.

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Lab products found in correlation

4 protocols using abc99

Notum Inhibition and Cell Proliferation in Mice

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ABC99 (Sigma-Aldrich, #SML2410) was prepared as previously described (59 (link)), except for the fact that a stock solution (16.5 mg/ml) was used. This stock was sequentially mixed with Tween 80 (Sigma-Aldrich, #P1754), polyethylene glycol, molecular weight 400 (Merck, #91893), and 0.9% NaCl in the ratio of 1:1:1:17. P45 Nfia/b/x triple knockout male mice were intraperitoneally injected with ABC99 (10 mg/kg) for five consecutive days. EdU (50 mg/kg of body weight) was injected together from the third day of treatment to profile proliferation induced by Notum inhibition (29 (link)). For the cyclopamine stock solution (1 μg/μl), cyclopamine (1 mg; Tocris Bioscience, #1623) powder was dissolved into 2-hydroxylpropyl-β-cyclodextrin (1 ml; Sigma-Aldrich, #C0926) prepared as a 45% solution in phosphate-buffered saline (PBS). cyclopamine (10 μg/g) was injected from P8 to P16 on alternative days. BrdU (100 μg/g of body weight) was injected together from the third injection for five consecutive days. Control mice were treated with the corresponding vehicle solution.

Yoo S., Kim J., Lyu P., Hoang T.V., Ma A., Trinh V., Dai W., Jiang L., Leavey P., Duncan L., Won J.K., Park S.H., Qian J., Brown S.P, & Blackshaw S. (2021). Control of neurogenic competence in mammalian hypothalamic tanycytes. Science Advances, 7(22), eabg3777.

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Protein Expression Analysis by Western Blot

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All proteins were extracted using RIPA-PMSF methods and quantified through BCA kit (Solarbio, Beijing, China). Briefly, 20 μg of loaded protein was separated with 7.5%, 10% or 12.5% SDS‒PAGE (Epizyme, Shanghai, China), and then transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated at 4°C overnight with the primary antibodies. The next day the membranes were incubated with the corresponding secondary antibody (Proteintech) for 2 h. Finally, the blots were visualized by Amersham Imager 600 system (GE, Boston, MA, USA). The relative quantitative values of the bands were measured by ImageJ software. Each assay was repeated three times.
Primary antibodies at 1:1000 dilution used for Western blotting were as follows: CD63 (ab134045; Abcam), TSG101 (ab125011; Abcam), PLAP (ab133602; Abcam), SPA (ab87674, Abcam; ab115791, Abcam), SPB (ab271345, Abcam; ab231551, Abcam), SPC (ABC99, Sigma-Aldrich; ab211326, Abcam), SPD (sc-25324, Santa Cruz Biotechnology; ab220422, Abcam), Sox9 (ab185966, Abcam), Bim (C34C5, CST), Bax (#2772, CST), Bcl2 (D17C4, CST), Cleaved Caspase-3 (1:500, 5A1E, CST) and tubulin (#2146, CST) as a control.

Chen P., Gu M., Wan S., Jiang X., Zhang F., Li Y., Zhou Q., Lu Y., Li L, & Wang X. (2023). Gestational Diabetes Mellitus Impedes Fetal Lung Development Through Exosome-Dependent Crosstalk Between Trophoblasts and Lung Epithelial Cells. International Journal of Nanomedicine, 18, 641-657.

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Immunostaining and Immunoblotting Techniques

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For immunostaining and immunoblotting, mouse monoclonal anti-LC3B (CTB-LC3-2-IC, Cosmo Bio), anti-ABCA3 (ab24751, Abcam), anti-HOPX (sc-398703, Santa Cruz Biotechnology), anti-HSP90 (610419, BD Transduction Laboratories), rat monoclonal anti-LAMP1 (ab25245, Abcam), and rabbit polyclonal anti-SPC (ABC99, EMD Millipore), anti-SPB (07-619, EMD Millipore), anti-p62 (PM045, MBL) antibodies were used as primary antibodies. Alexa Fluor 488-conjugated anti-mouse IgG (A11001, Thermo Fisher Scientific), Alexa Fluor 568-conjugated anti-rabbit IgG (A11011, Thermo Fisher Scientific), Alexa Fluor 568-conjugated anti-ratbbit IgG (A11077, Thermo Fisher Scientific), Alexa Fluor 633-conjugated anti-rat IgG (A11094, Thermo Fisher Scientific), Alexa Fluor 660-conjugated anti-mouse IgG (A-21055, Thermo Fisher Scientific), Alexa Fluor 660-conjugated anti-rabbit IgG (A21074, Thermo Fisher Scientific), HRP-conjugated goat polyclonal anti-rabbit IgG (111-035-144, Jackson ImmunoResearch Laboratories), and HRP-conjugated goat polyclonal anti-mouse IgG (115-035-003, Jackson ImmunoResearch Laboratories) antibodies were used as secondary antibodies. Hoechst33342 (H342, Dojindo Molecular Technologies) was used to stain DNA. Bafilomycin A₁ (B1793) was purchased from Sigma-Aldrich.

Morishita H., Kanda Y., Kaizuka T., Chino H., Nakao K., Miki Y., Taketomi Y., Guan J.L., Murakami M., Aiba A, & Mizushima N. (2020). Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder. Cell reports, 33(10), 108477.

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Quantifying AEC2 Purity in Lung Cells

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To quantify the AEC2 purity in MACS-isolated CD45(-)EpCAM(+) populations, we performed immunofluorescent staining of surfactant protein C (SP-C) using cytospin slides. After isolation, the CD45(-)EpCAM(+) cells were fixed by 4% PFA in a flow cytometry tube for 12 min at room temperature, and were transferred to slides by cytospin centrifugation at 13.83 × g (350 r.p.m.) for 3 min. The CD45(−)EpCAM(−) population was used to prepare cytospin slides for negative control. Blocking and permeabilization were performed at room temperature for 1 h, using a buffer containing 5% normal goat serum (Vector Laboratories) and 0.3% Triton X-100 (Sigma-Aldrich) in tris-buffered saline (TBS). Cells were incubated overnight with a primary antibody generated to SP-C (1:1000 in blocking buffer, EMD Millipore ABC99) in a humidified chamber at 4 °C. Sixteen to 24 h later, the cells were incubated with the Alexa Fluor-488-conjugated secondary antibody (Thermo Fisher) for 1 h at room temperature. Hoechst 33342 (1:1000 dilution in TBS) was used to stain the nucleus. The slides were mounted using Prolong Gold antifade solution (Invitrogen), and the images of the slides were obtained by confocal microscopy (Zeiss LSM 880 laser scanning microscope).

Chung K.P., Hsu C.L., Fan L.C., Huang Z., Bhatia D., Chen Y.J., Hisata S., Cho S.J., Nakahira K., Imamura M., Choi M.E., Yu C.J., Cloonan S.M, & Choi A.M. (2019). Mitofusins regulate lipid metabolism to mediate the development of lung fibrosis. Nature Communications, 10, 3390.

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