Immunofluorescences (IF) and immunoblots (WB) were performed with the following primary antibodies: anti-rabbit S100A4 (1:500-IF, 1:1000-WB, Millipore), anti-rabbit mTOR and phospho-mTOR (1:100-WB, Cell Signaling), anti-mouse SQSTM1/p62 (1:1000-WB, Abcam), anti-rabbit NF-kB and phospho-NF-kB (1:1000-WB, Cell Signaling), anti-rabbit STAT3 (1:1000-WB, Cell Signaling), anti-rabbit phospho-STAT3 (1:2000-WB, Cell Signaling), anti-rabbit α-SMA (1:1000-WB, 1:500-IF, GeneTex), anti-rabbit N-cadherin (1:1000-WB, GeneTex), anti-mouse glial fibrillary acidic protein (GFAP) (1:500-IF, 1:1000-WB, Cell Signaling), anti-rabbit β-III Tubulin (1:500-IF, Cell Signaling), anti-mouse MyoG (1:200-WB, Hybridoma Bank, USA), anti-mouse platelet-derived growth factor receptor β (PDGFR-β) (1:250-WB, 1:500-IF, Santa Cruz), and anti-mouse GAPDH (1:10000-WB, Calbiochem). Secondary immunoblot antibodies for WB were anti-rabbit (1:2500) and anti-mouse (1:5000) IgG peroxidase-conjugated from Bio-Rad Laboratories (Hercules, CA, USA). Secondary fluorescent antibodies for IF were Alexa-Flour 488-Donkey anti-rabbit (1:200) and Cy3-Donkey anti-mouse (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI was used to stain nuclei (1:1000, Thermo Fisher Scientific, Waltham, MA, USA).
Anti mouse
Manufactured by Bio-Rad
Sourced in United States
The Anti-mouse product is a laboratory reagent used for the detection and analysis of mouse-derived biological samples or components. It functions as a specific binding agent to identify and quantify the presence of mouse-derived proteins, cells, or other biomolecules in experimental or diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit, by Bio-Rad (17 mentions)
Image lab software, by Bio-Rad (5 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Anti rabbit, by GE Healthcare (3 mentions)
Anti rabbit, by Cell Signaling Technology (3 mentions)
Mlph antibody, by Proteintech (3 mentions)
Horseradish peroxidase conjugated anti rabbit, by Fortis Life Sciences (3 mentions)
β actin antibody, by Merck Group (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Hrp conjugated anti rabbit antibody, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti rabbit, by Merck Group (2 mentions)
Myova antibody, by Cell Signaling Technology (2 mentions)
Rab27a antibody, by Santa Cruz Biotechnology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
56 protocols using anti mouse
1
Immunofluorescence and Immunoblotting Analysis
2
Multiplex Immunohistochemistry Assay for Immune Markers
3
Evaluating B Cell Subsets in Sows
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To assess the adaptive immune response in the sows, frequencies of B lymphocyte subpopulations were evaluated using a previously established protocol [45 (link)]. Briefly, to identify selected B cell subsets, isolated MNCs were stained with anti-porcine CD21-PE (clone BB6-11C9.6, Southern Biotech, Birmingham, AL, USA), anti-porcine CD2 (clone MSA4, King Fisher Biotech, St Paul, MN, USA), and anti-mouse (porcine cross-reactive) CD79β-FITC (clone AT1072, Bio-Rad, Hercules, CA, USA) mAbs. Additionally, mouse anti-pig IgA mAb (K61 1B4, Bio-Rad, Hercules, CA, USA) was used to evaluate the frequencies of IgA producing B cells among the MNCs. Finally, human (porcine cross-reactive) integrin alpha 4 beta 7(α4β7)/LPAM-1 (Clone # Hu117, R&D Systems, Minneapolis, MN, USA) and anti-mouse CCR10 (clone 248918, R&D Systems, Minneapolis, MN, USA) were used to examine the expression of gut homing markers (α4β7/CCR10) among the isolated MNCs. Suitable isotype control and secondary Abs were included in each assay. Stained samples were analyzed using Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) with acquisition set at 50,000 events. The frequencies of different cell populations were determined using CFlow software (AccuriC6 cytometers; BD Biosciences, San Jose, CA, USA).
4
Western Blot Analysis of Protein Abundance
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Cells were lysed with EBC lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM Complete protease inhibitors (Roche Diagnostics, Basel, Switzerland, catalog # 11697498001) and 1 mM phosphatase inhibitor cocktail III (Sigma-Aldrich, St. Louis, MO, USA, catalog # P0044)). SDS-PAGE was done followed by transferring the proteins onto nitrocellulose membranes, and blocking the membranes with 5% non-fat milk in phosphate-buffered saline with tween 20 (PBS-T) for 30 min. Incubation with the primary antibodies was done overnight at 4 °C followed by 1-h incubation with the appropriate secondary antibodies at room temperature. The following primary antibodies were used: anti-ERK3 (Abcam, Cambridge, United Kingdom, catalog # ab53277), anti-phospho-ERK3 (S189), generated by our lab as described previously [9 (link)], anti-septin 7 (Immuno-Biological Laboratories, Minneapolis, MN, USA, catalog # 18991), and anti-β-actin (Sigma-Aldrich, catalog # A5316), and the following secondary antibodies were used: anti-mouse (Biorad, Hercules, CA, USA, catalog # 170-6516) and anti-rabbit (Biorad, catalog # 170-6515). The Western blots were visualized by chemiluminescence (ThermoFisher, catalog # 32109). β-actin was used as a loading control.
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed in an ice-cold RIPA buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl,1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1× protease inhibitors (cOmplete mini EDTA-free, Roche) and 1× phosphatase inhibitors (PhosStop, Roche)] and protein concentrations of the lysates were determined by Pierce Micro BCA assay (cat. 22225). Equal total protein amount was loaded into stacking and resolving SDS–polyacrylamide gel electrophoresis (PAGE) gel. Using a wet transfer system, proteins were transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk in 1× TBST buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Primary antibodies were incubated at 4°C overnight [CTCF (BD, 1:1000, cat. 612149), GAPDH (Cell Signaling, 1:10 000, cat. 5174), p53 (Santa Cruz, 1:500, cat. sc-126) and γH2AX (Ser139) (Abcam, 1:5000, cat. ab11174)]. Membranes were washed and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse (Bio-Rad, 1:500, cat. 170-5047) and anti-rabbit (Bio-Rad, 1:500, cat. 170-5046), respectively. Pierce™ ECL (cat. 32109) kit was used for HRP substrate detection. Images were captured by Bio-Rad ChemiDoc Imaging System and signal intensity was quantified by Bio-Rad Image Lab Software.
6
Western Blot Analysis for Protein Detection
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Western blot analyses were carried out as described previously [23] (link). In brief, lysates from NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were collected and 100 µg of total protein was loaded onto a 7.5% or 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis and electroblotting onto a nitrocellulose membrane, proteins were detected with primary antibodies against human HIF-1α (1:2000; BD Bioscience), mouse HIF-1α (1:1000; Novus Biologicals), MnSOD (SOD2) (1:1000; Calbiochem), MECR (1:1000; Proteintech), and against α-tubulin (1:10.000; Sigma). The secondary antibody was either an anti-mouse, an anti-rabbit or an anti-sheep immunoglobulin G conjugated to horseradish peroxidase (1:5000; Bio-Rad Laboratories). The ECL system (Amersham) was used for detection.
For half-life studies, NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were cultured under normoxic or hypoxic conditions. After 20 h, cycloheximide (10 µg/ml; Sigma) was added to the cell culture medium, cells were scraped in lysis buffer (50 mM Tris/HCL, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF and complete protease inhibitor cocktail tablet (Roche)) at indicated time points and protein levels were measured by immunoblot analysis.
For half-life studies, NIH 3T3-scr, NIH 3T3-MECR-KD, HepG2-scr, HepG2-MnSOD-KD, Mpv17+/+ and Mpv17-/- cells were cultured under normoxic or hypoxic conditions. After 20 h, cycloheximide (10 µg/ml; Sigma) was added to the cell culture medium, cells were scraped in lysis buffer (50 mM Tris/HCL, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF and complete protease inhibitor cocktail tablet (Roche)) at indicated time points and protein levels were measured by immunoblot analysis.
7
Western Blot and Immunoprecipitation Protocols
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For immunoblotting, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, UK). Blots were incubated for 30 min in Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dry milk and further incubated for two hours (or overnight at 4° C) with the following antibodies: rabbit anti-ADAM12 (Sigma), rabbit anti-ZO-1 (Life Technologies). The bound antibodies were detected with horseradish peroxidase-conjugated to either anti rabbit (Pierce) or anti-mouse (BioRad, Ivry, France) IgG or Protein A using an enhanced chemiluminescence system (Millipore, Billerica, MA, USA).
For immunoprecipitation, cell extracts were prepared in RIPA buffer and pre- incubated for 1 hour with sepharose-coupled protein-G beads (Amersham) alone to reduce non specific protein binding and then with sepharose-coupled protein G prebound with 1μg of either specific antibodies or control rabbit IgG. The beads were washed five times in buffer and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.
For immunoprecipitation, cell extracts were prepared in RIPA buffer and pre- incubated for 1 hour with sepharose-coupled protein-G beads (Amersham) alone to reduce non specific protein binding and then with sepharose-coupled protein G prebound with 1μg of either specific antibodies or control rabbit IgG. The beads were washed five times in buffer and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.
8
Protein Quantification and Western Blot
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Purified protein lysates were quantified with a bicinchoninic acid (BCA) assay (ThermoFisher). For SDS-PAGE, 20 μg/sample of prepared protein (cell lysate with 1X LDS sample buffer) were run on a 4–12% Bis-Tris polyacrylamide gel at 120 V for 60 min in 1X running buffer (2.5 mM MOPS, 2.5 mM Tris base, 0.005% SDS, 50 μM EDTA, pH 7.7). Protein was transferred to PVDF membrane at 30 V for 960 min in 1X transfer buffer (1.25 mM Bicine, 1.25 mM Bis-Tris, 50 μM EDTA, pH 7.2). Membranes were blocked in Starting Block T20 (ThermoFisher). Primary antibodies were used at a 1:2000 dilution (MME (human: Santa Cruz, mouse: Abcam), IRα (Santa Cruz), IRβ (Cell Signaling), pIR/pIGF1R (Cell Signaling), IRS1 (Millipore), pIRS1 (Cell Signaling), pan-AKT (Cell Signaling), pAKT (Cell Signaling), ERK1/2 (Cell Signaling), pERK1/2 (Cell Signaling), Actin (Santa Cruz), GAPDH (Santa Cruz)). Secondary antibodies were used at a 1:10,000 dilution (anti-mouse (Bio-Rad) or anti-rabbit (Bio-Rad)). Signal was detected by Pico or Femto Chemiluminescent Substrate (ThermoFisher).
9
Western Blot Analysis of Immune Sensors
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Protein extraction was performed by lysing the cells in Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels and electrotransferred to nitrocellulose membranes (Bio-Rad, Cat. No. 162-0115). Non-specific binding sites were blocked with 5% non-fat dry milk diluted in TBS Tween buffer (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4). The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778). The bound antibodies were labeled with anti-mouse (Bio-Rad, Cat. No. 1721011), anti-rat (Bio-Rad, Cat. No. 5204-2504) or anti-rabbit (GE Healthcare, Cat. No. NA934) horseradish peroxidase-conjugated secondary antibodies and were visualized by the ECL system using SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Scientific, Rockford, IL, USA, Cat. No. 34580 and 34095) and X-ray film exposure. Densitometric analysis of immunoreactive bands was performed using Image Studio Lite Software version 5.2 (LI-COR Biosciences, Lincoln, Nebraska USA).
10
Protein Expression Analysis of Brain Microvessels
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Isolated brain microvessels were washed with PBS and then lysed in RIPA buffer. Western blotting was performed with anti-claudin-5, -ZO-1, -IL6, -β-actin, and -GADPH antibodies (Cell Signaling Technology, Danvers, MA, United States). Immunoblots were exposed to secondary antibodies, either anti-mouse- or anti-rabbit-HRP conjugated antibody (BioRad), and visualized with a chemiluminescent HRP substrate kit and analyzed using Image J software.
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