Cells were harvested in lysis solution containing 50 mM Tris/HCl (pH 7.6), 1% NP40, 150 mM NaCl, 2 mM EDTA, 100 μM PMSF, a protease inhibitor cocktail (Roche Applied Science), and a phosphatase inhibitor (Sigma-Aldrich). After incubation on ice for 30 min, cellular debris was removed by centrifugation (10 min, 4 °C). Proteins (10 μg) were separated by SDS–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk, the membranes were probed with an appropriate antibody. Blots were developed with an enhanced chemiluminescence western blotting detection system (Amersham, GE Healthcare, Buckinghamshire, UK). The following antibodies were used: anti-β-actin (Sigma-Aldrich) and anti-mouse albumin (Santa Cruz Biotechnology).
Mouse anti albumin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-albumin is a laboratory reagent used for the detection and quantification of albumin, a common protein found in the blood. It is a primary antibody that specifically binds to the albumin molecule, allowing for its identification and measurement in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Rabbit anti crp, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence western blotting detection system, by GE Healthcare (1 mentions)
A phosphatase inhibitor, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Confocal fluorescence microscope, by Olympus (1 mentions)
Mouse anti gata4, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fv10 asw 2.0 viewer, by Olympus (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Rabbit anti β actin, by Merck Group (1 mentions)
Clarity western ecl kit, by Bio-Rad (1 mentions)
Anti mouse vegf, by R&D Systems (1 mentions)
Anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa 488 and 568, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Methanol, by Merck Group (1 mentions)
6 protocols using mouse anti albumin
1
Western Blot Protein Extraction Protocol
2
Immunofluorescence Staining of Differentiated Cells
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Cells at each differentiation stage were fixed with 4% paraformaldehyde and stained with the appropriate antibody. The following antibodies were used: anti-mouse Foxa2 (Abcam, Cambridge, MA, USA), anti-mouse Gata4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse Trop2 (R&D Systems), A6 antibody (kindly given by Dr Valentina Factor), anti-mouse albumin (Santa Cruz Biotechnology), Alexa 546-conjugated donkey anti-rabbit IgG (Invitrogen Life Technologies, Carlsbad, CA, USA), Alexa 647-conjugated donkey anti-goat IgG (Invitrogen) and Alexa 488-conjugated donkey anti-mouse IgG (Invitrogen). Image acquisition and processing was performed using a confocal fluorescence microscope (Olympus, Center Valley, PA, USA) and an FV10-ASW 2.0 Viewer (Olympus).
3
Western Blot Analysis of Liver Proteins
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Cells and liver tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 ug protein extracts were separated in 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membrane was incubated with rabbit anti-CRP (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-albumin (1:1,000 Santa Cruz), mouse anti-VEGF, rabbit anti-VEGFR1, mouse anti-endoglin (1:1,000 R&D Systems, Abingdon, UK), and rabbit anti-β-actin (1:3,000, Sigma Aldrich) at 4℃ overnight. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG [1:20,000, Bio-Rad Laboratories, Hercules, CA, USA] or antimouse IgG [1:5,000, Santa Cruz]) for 1 hour at room temperature. The bands were detected using Clarity Western ECL kit (Bio-Rad).
4
Localization of C-Reactive Protein in Liver
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To analyze the localization of CRP in the liver tissues, frozen liver sections were treated with the Blocking Solution (DAKO, Glostrup, Produktionsvej, Denmark) at room temperature for 40 minutes, and rabbit anti-CRP (1:50, Santa Cruz) was applied to the sections at 4℃ overnight. Samples were washed with PBS, and incubated with Alexa Fluor 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. To detect the co-localization of albumin and CRP in WB-F344 cells incubated with siRNA-CRP and LCA, WB-F344 cells were fixed with 100% methanol (Merck, NJ, USA). The cells were reacted with Blocking Solution (Dako) for 1 hour at room temperature and mouse antialbumin (1:50, Santa Cruz) and rabbit anti-CRP (1:50, Santa Cruz) at 4℃ overnight. After reaction, the cells were incubated with Alexa 488 and 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. The slides were stained with 4’, 6-diamidino-2-phenylindole (DAPI). The images were observed with a fluorescence microscope (Nikon, Tokyo, Minato, Japan). All experiments were performed in triplicate.
5
Immunofluorescence Analysis of Spinal Cord Pathology
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Immunofluorescence analysis was accomplished as previously described [20 (link)–22 (link)]. Briefly, cryosections (10 μm thick; n = 3 per spinal cord; 5 spinal cords per group) from lumbar spinal cords in each group were incubated with rat anti-CD3 (1:500; BD Biosciences, NJ, USA), rat anti-myelin basic protein (MBP) (1:1000; Abcam), anti-ionized calcium binding adaptor molecule-1 (Iba-1) antibody (1:2000; Wako, Osaka, Japan), mouse anti-glial fibrillary acidic protein (GFAP; 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and/or rat anti-platelet endothelial cell adhesion molecule (PECAM)-1 (1:500; Santa cruz), mouse anti-albumin (1:500; Santa cruz), and rabbit anti-immunoglobulin G (IgG) (1:500; Abcam), mouse anti-occludin (1:500; Invitrogen, MA, USA), mouse anti-ZO-1 (1:500; Invitrogen) as primary antiserum and cyanine 3- and fluorescein-isothiocyanate (FITC)-conjugated mouse/rabbit/rat IgG antibody (1:200–1:500; Jackson ImmunoResearch, West Grove, PA, USA) as secondary antiserum. Images from each section were captured using confocal imaging system (LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany) and their intensity quantified. Immunohistochemical analysis for Iba-1 was accomplished and analyzed as previously described [20 (link)–22 (link)].
6
Immunofluorescence Analysis of Islet Cells
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Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After antibody blocking, primary antibodies were incubated overnight at 4 °C. We used rabbit anti-insulin and mouse anti-glucagon (1:1000; Abcam, Cambridge, UK), rabbit anti-PDX1, goat anti-PDX1, mouse anti-NEUROD1, rabbit anti-MAFA (1:200; Abcam), and mouse anti-albumin (1:100, Santa Cruz Biotechnology). For secondary fluorescence labeling, the cells were incubated with anti-rabbit IgG Alexa Fluor 488, anti-rabbit IgG Alex Fluor 555, anti-goat IgG Alex Fluor 488, anti-mouse Alexa 555, and anti-mouse Alexa 488 (1:200; Thermo Fisher Scientific). ProLong Gold antifade reagent containing DAPI (Thermo Fisher Scientific) was used to stain the nuclei and for mounting. The slides were visualized using the EVOS® FL auto cell imaging system (Thermo Fisher Scientific). Under the microscope, the cells with red fluorescence were insulin or glucagon positive and blue fluorescence highlighted the nucleus and counted in 10 randomly selected fields per IPC sheet or IPC cells with total of 3 donors in each group. The results were presented as insulin- or glucagon-positive cells per 100 cells.
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