General cell morphology was visualized after fixation with 4% paraformaldehyde through staining with 4% Giemsa solution for 1 min, washing with distilled water and natural drying. For cytoskeleton β-actin immunocytochemistry, after fixation with 4% paraformaldehyde, cells were permeabilized with 100% methanol at −20 °C for 15 min, and 0.5% Triton X-100 (Amresco Inc., Solon, OH, USA) in PBS for 15 min. Non-specific binding was blocked with 1% goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. After each incubation step, the samples were washed thrice under gentle agitation on a horizontal shaker table for 5 min. The primary antibodies anti β-actin (mouse monoclonal anti βACT antibody, working dilution of 1:1000, MA1-140, Thermo Fisher Scientific, Rockford, IL, USA) were incubated overnight under refrigeration and were followed by corresponding goat anti-mouse (IgG FITC, working dilution of 1:1000, Sigma Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Hoechst’s solution (H3569, 2μL/mL for 5 min, Invitrogen, Eugene, OR, USA) was used for nuclear staining. Cultures were examined under a phase contrast microscope (Zeiss Axio Vert.A1, Zeiss, Jena, Thuringia Land, Germany), equipped with an AxioCam MRC camera (Zeiss, Jena, Thuringia Land, Germany).
Ma1 140
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MA1-140 is a laboratory equipment product offered by Thermo Fisher Scientific. It serves as a device for performing specific laboratory functions, but a detailed and unbiased description cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Axio vert a1, by Zeiss (1 mentions)
Axiocam mrc camera, by Zeiss (1 mentions)
Goat anti mouse igg fitc, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti mouse a4416, by Merck Group (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Enhanced chemiluminescence, by Solarbio (1 mentions)
Np0302box, by Thermo Fisher Scientific (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence reagent, by Bio-Rad (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
5 protocols using ma1 140
1
Cell Morphology and Cytoskeleton Visualization
2
Biochemical Fractionation and Immunoblotting of α-Synuclein
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Cortex and hippocampi from 4 week-old wildtype C57BL/6J mice and Rab27 DKO mice were dissected, homogenized with a motorized tissue grinder, and sonicated in lysis buffer (175 mM NaCl, 50 mM Tris–HCl, pH 7.4, 5 mM EDTA, protease inhibitor cocktail [ThermoFisher Scientific]). After 1% Triton X-100 was added, and after a 30 min incubation on ice, samples were centrifuged at 15,000g × 60 min at 4 °C. Supernatant was the Triton X-100 soluble fraction. Pellets were resuspended in lysis buffer with 2% SDS, sonicated for 10 s, and then spun at 15,000g for 10 min. Supernatant was the Triton X-100 insoluble fraction. Western blot analysis was performed as described previously45 (link). Briefly, equal total protein was loaded per sample, resolved on a 12% SDS–polyacrylamide gel, and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk solution followed by incubation in primary antibody (αsyn antibodies: Becton Dickinson #610787 and Cell Signaling Technology #2642S; β-actin antibody ThermoFisher Scientific MA1-140) at 4 °C overnight. Membranes were then incubated in HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch) and developed with the enhanced chemiluminescence method. Images were scanned using the Bio-Rad Chemidoc Imaging System.
3
Everolimus and PEG-LIP(ev)-HA400kDa Effect on mTOR Pathway
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LFs were seeded on 6-well plate at a density of 5 × 105 cells and after 24 h were incubated with PEG-LIP(ev), PEG-LIP(ev)-HA400kDa and everolimus alone, with the same concentration of everolimus (50 nM). After 24 h, cells were washed with PBS, lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to PVDF membranes (Millipore), therefore blocked (5% BSA in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 5% BSA; overnight at 4 °C): anti-mTOR (1:1000) (PA1518—abcam), anti-p-mTOR(Ser2448) (1:1000) (PA585736—abcam), and anti-β-Actin (1:5000) (MA1-140—Thermo Fisher Scientific). After wash, the membranes were incubated with the appropriate horseradish-peroxidase conjugated secondary Ab (1:5000 in TBST + 5% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (BioRad, Segrate, Italy), acquired with the Uvitec alliance mini H9 (Uvitec Ltd, Cambridge, UK).
4
Quantifying Colon Protein Expression
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Colon samples were homogenized using a protein extraction kit, in which the phenylmethanesulfonyl fluoride, protease inhibitors, and phosphatase inhibitors were mixed (Solarbio Life Sciences, Beijing, China), and then centrifuged at 12,000g for 20 min at 4°C. The protein concentration was determined using a bicinchoninic acid protein determination kit (Yeasen Technologies, Shanghai, China). For Western blot analysis, 50 μg of protein extract was separated with 10% NuPAGE (NP0302BOX, Invitrogen) and then transferred to a polyvinylidene fluoride (PVDF) membrane. This was sealed with 5% skim milk for 1 h at 28°C, using anti-p65 (51-0500, Invitrogen), anti-IL-1β (MM425B, Invitrogen), anti-TNF-α (AMC3012, Invitrogen), anti-IκBα (MA5-16152, Invitrogen), anti-Cox-2 (PA5-17614, Invitrogen), and anti-β-actin (MA1-140, Invitrogen); incubated in the PVDF membrane; washed five times with 1× TBST; and then combined with Horseradish peroxidase (HRP) secondary antibodies (A32723, Invitrogen). Next, the mixture was incubated for 1 h at room temperature and the PVDF membrane was washed five times with 1× TBST. Antibody binding was observed using enhanced chemiluminescence (Solarbio Life Sciences, Beijing, China). Finally, ImageJ software (U.S. National Institutes of Health, Bethesda, MD, United States) was used to quantify protein expression.
5
Western Blot Analysis of VEGF
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Retinal samples were lysed with cock tail of radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor (Sigma, St Louis, MO), and total protein concentration was estimated by a BCA protein assay kit (23227, Pierce Thermo Fisher). An equal amount of protein (15 µg) from each group was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF, Bio-Rad Laboratories). The primary antibody VEGF (MA1-16629, Invitrogen), beta-actin (MA1-140, Invitrogen), and appropriate secondary antibodies were used. The protein was detected with a chemiluminescence reagent (Bio-Rad Laboratories) and hydrogen peroxide. beta-actin from the same immunoblot was a loading control, and band intensity was analyzed with ImageJ software.
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