Fluorchem 9900

Manufactured by Bio-Techne

Sourced in United States

The FluorChem 9900 is a high-performance imaging system designed for the visualization and analysis of fluorescent samples. It offers advanced imaging capabilities, including digital image capture, quantitative analysis, and comprehensive data management solutions.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) Rnalater, by Merck Group (1 mentions) Ab37073, by Abcam (1 mentions) Ab66579, by Abcam (1 mentions) Sc 23948, by Abcam (1 mentions) Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions) Luminata forte, by Merck Group (1 mentions) Ab54741, by Abcam (1 mentions) Ab77302, by Abcam (1 mentions) Enhanced chemiluminescence kit, by Merck Group (1 mentions) Ab205718, by Abcam (1 mentions)

4 protocols using fluorchem 9900

Quantitative mRNA Expression Analysis

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mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR). Pancreatic samples were rapidly immersed in RNAlater (Sigma-Aldrich Co.) for RNA protection and stored at −20°C before assay. Total RNA was extracted from pancreatic tissues using a TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and was reverse-transcribed using oligo (dT) as a primer. The sequences used as an internal standard control, housekeeping gene β-actin, and target genes are listed in Table 1.
PCR amplification cycles were carried out under the following conditions: initial activation of 95°C for 5 minutes followed by 35 cycles of 45 seconds of denaturation at 95°C, 45 seconds of annealing at (58°C for β-actin, ICAM-1, and α-SMA; 60°C for NF-κB/p65; 50°C for TNF-α), 45 seconds of extension at 72°C followed by one final extension at 72°C for 7 minutes. Completed reactions were held at 4°C.
PCR products were separated by gel electrophoresis (1.5% agarose stained with ethidium bromide). Specific bands were visualized with an image system (FluorChem 9900; Alpha Innotech, San Leandro, CA, USA). The detection of each gene was repeated for three times. The optical density (OD) values of the bands were analyzed using ImageJ software and standardized to the β-actin signal.

Wang Y.R., Tian F.L., Yan M.X., Fan J.H., Wang L.Y., Kuang R.G, & Li Y.Q. (2016). Sulfasalazine inhibits inflammation and fibrogenesis in pancreas via NF-κB signaling pathway in rats with oxidative stress-induced pancreatic injury. Drug Design, Development and Therapy, 10, 1743-1751.

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Hypothalamic ER Stress Signaling Proteins

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A subset of 6 mice per group were used for assessment of hypothalamic PERK, pPERK^Thr980, eIF2α, peIF2α^Ser55, IRE1α, pIRE1α^Ser724, ATF6, TNFα and tubulin protein expression using standard methodology, as previously described (McGavigan et al., 2015 (link)). Antibodies against PERK, pPERK^Thr980 and peIF2α^Ser55 were from Cell Signaling (3179 and 3398, respectively). Antibodies against eIF2α, ATF6 and tubulin were from Santa Cruz (sc-133227, sc-22799, sc-23948, respectively) and TNFα, IRE1α and pIRE1α^Ser724 antibodies were from Abcam (ab66579, ab37073, ab104157, respectively). All antibodies were used at a concentration of 1:1000 and pixel densities of immunoreactive bands were quantified using FluorChem 9900 (Alpha Innotech, CA).

McGavigan A.K., Henseler Z.M., Garibay D., Butler S.D., Jayasinghe S., Ley R.E., Davisson R.L, & Cummings B.P. (2017). Vertical sleeve gastrectomy reduces blood pressure and hypothalamic endoplasmic reticulum stress in mice. Disease Models & Mechanisms, 10(3), 235-243.

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Mouse Tissue Proteomics Analysis

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Mouse tissues were dissected and immediately frozen in liquid nitrogen. For zymography, liver samples were homogenized in cold 25 mM Tris-HCl buffer using 3 × 30 s bursts of a roto-stator homogenizer with one minute intervals in between. Zymography was performed using Novagen pre-cast gelatin zymography gels (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions and quantified using ImageJ (NIH). For Western blots, tissues were ground in the presence of liquid nitrogen and lysed using radio-immunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate and protease inhibitors). Lysates were clarified by centrifugation at 10,000 × g for 10 min and protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA). Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. Immunoblots were performed with the relevant antibodies. Proteins were visualized using Luminata^™ Forte (Millipore, Billerica, MA). For quantitation purposes, pixel intensities of immuno-reactive bands from blots that were in the linear range of loading and exposure were quantified using FluorChem 9900 (Alpha Innotech, San Lenardo, CA).

Harris T.R., Bettaieb A., Kodani S., Dong H., Myers R., Chiamvimonvat N., Haj F.G, & Hammock B.D. (2015). Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice. Toxicology and applied pharmacology, 286(2), 102-111.

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HMGB1 and RAGE Protein Detection

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Total proteins were extracted from the cells via treatment with lysis buffer and benzylsulfonyl fluoride (PMSF) (both from Beyotime, Shanghai, China), and then the protein supernatants were centrifuged at 10000 rpm for 10 min (at 4°C) to remove tissue fragments from sediments. The precipitate was then quantified using a bicinchoninic acid protein assay. Next, protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membrane was blocked with 5% fat-free milk for 2 hours, and incubated with either anti-HMGB1 antibody (ab77302, 1:1000 dilution, Abcam, Cambridge, MA, USA) or anti-RAGE antibody (ab54741, 1:200 dilution, Abcam) at 4°C, overnight. The blots were incubated with horseradish peroxidase-conjugated secondary antibodies (ab205718, 1:2000 dilution, Abcam) at room temperature (25°C) for 1 hour with continuous shaking. After washing, proteins within Western blots were detected using an enhanced chemiluminescence kit (Millipore, MA, USA), and then the blots were exposed to an X-ray film. Bands were quantified using Fluorchem (9900) (Alpha Innotech, Sam Leandro, CA, USA) equipment, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

Liu C., Sun H., Tang M., Li J., Zhang X, & Cao G. (2021). Ethyl Pyruvate Alleviates Pulmonary Hypertension through the Suppression of Pulmonary Artery Smooth Muscle Cell Proliferation via the High Mobility Group Protein B1/Receptor for Advanced Glycation End-Products Axis. Annals of Thoracic and Cardiovascular Surgery, 27(6), 380-388.

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