Yeast whole-cell extracts were prepared, normalized for protein concentration and analyzed by western blotting according to standard procedures. Antibodies were used according to the manufacturer’s instructions: mouse monoclonal anti-mCherry (Abcam); mouse monoclonal anti-GFP (clones 7.1 and 13.1) (Roche); mouse monoclonal anti-Pgk1 (Abcam); peroxidase AffiniPure Goat anti-Mouse IgG (Jackson ImmunoResearch).
Affinipure goat anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
AffiniPure goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG). It is designed for use in various immunoassays and other applications requiring the detection of mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Ecl system, by CWBIO (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Affinipure goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Mouse monoclonal anti pgk1, by Abcam (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Mini protean 3, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscopy, by Leica (1 mentions)
Nunc lab tek, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Ripa reagent, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 aobs, by Leica (1 mentions)
β catenin 610153, by BD (1 mentions)
β catenin, by Jackson ImmunoResearch (1 mentions)
Hydromount hs 106, by National Diagnostics (1 mentions)
Bx61 microscope, by Leica (1 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate, by Merck Group (1 mentions)
28 protocols using affinipure goat anti mouse igg
1
Western Blot Analysis of Yeast Proteins
2
Western Blot Analysis of Viral Proteins
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Proteins were denatured in boiling water for 10 min and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 5% stacking gel and 12% running gel) with a MINI-PROTEAN III apparatus (Bio-Rad; Hercules, CA, USA) [47 (link)]. Proteins resolved in SDS-PAGE were electrophoretically transferred to PVDF membranes (GE Healthcare Life Sciences, München, Germany). The membranes were incubated for 1 h at room temperature in PBS containing 0.1% Tween-20 and 5% dried milk. After removing the milk, the membrane was incubated with the primary antibody, i.e., mouse anti-nucleocapsid protein (1:4000 dilution) or anti-β-actin (1:1500 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 10-fold diluted PRO Blocking Buffer (Energenesis Biomedical Inc., Taipei, Taiwan) at 4 °C overnight. The following day, the membranes were incubated with the secondary antibody (1:5000 dilution of peroxidase-conjugated AffiniPure goat anti-mouse IgG; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h. The signal was visualized with the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Madrid, Spain) after rinsing with PBS containing 0.1% Tween-20.
3
Regulation of MUC5AC in LS174T Cells
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LS174T cells (60,000) were growth in each well of an 8-wells Nunc Lab-Tek (Thermo Scientific) at 37°C for 48 h in a humidified atmosphere with 5% CO2. Washed cells, were kept for 2 h in DMEM medium without FBS and antibiotics. Then, washed cells were replaced with 300 µl of fresh DMEM medium without supplements and incubated with 5 µg/ml of Pic, PicS258I or PMSF-preincubated Pic, or 0.125 mM DCA at 37°C for 4 h with 5% CO2.
After incubation time, culture medium was removed and cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were not permeabilized but blocked with 3% BSA for 30 min. Washed cells were incubated with 120 µl of the monoclonal anti-MUC5AC antibody 45M1 (1:100) at 37°C for 3 h. Cells were washed seven times and incubated 1 h with Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG (1:800, Jackson ImmunoResearch Labs, West Grove, PA) secondary antibody followed by dichlorotriazinylamino fluorescein (DTAF)-conjugated streptavidin (1:800, Jackson ImmunoResearch Labs). Finally, cells were stained with Rhodamine Phalloidin (1:80, Invitrogen) to stain F-actin and TO-PRO-3 (1:400, Invitrogen) to stain nuclei. Then, slides were washed and mounted using VECTASHIELD (Vector Laboratories, Burlingame, CA) antifade mounting medium and analyzed using a Leica confocal microscopy TCS SP8 (Leica Microsystems, Wetzlar, Germany).
After incubation time, culture medium was removed and cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were not permeabilized but blocked with 3% BSA for 30 min. Washed cells were incubated with 120 µl of the monoclonal anti-MUC5AC antibody 45M1 (1:100) at 37°C for 3 h. Cells were washed seven times and incubated 1 h with Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG (1:800, Jackson ImmunoResearch Labs, West Grove, PA) secondary antibody followed by dichlorotriazinylamino fluorescein (DTAF)-conjugated streptavidin (1:800, Jackson ImmunoResearch Labs). Finally, cells were stained with Rhodamine Phalloidin (1:80, Invitrogen) to stain F-actin and TO-PRO-3 (1:400, Invitrogen) to stain nuclei. Then, slides were washed and mounted using VECTASHIELD (Vector Laboratories, Burlingame, CA) antifade mounting medium and analyzed using a Leica confocal microscopy TCS SP8 (Leica Microsystems, Wetzlar, Germany).
4
ELISA for Antibody Quantification
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Concentration of antibodies against Gag and Min in mouse sera samples was determined by ELISA. Nunc MaxiSorp 96-well plates (ThermoFisher Scientific) were coated with either 50 ng of recombinant Gag or MinTT in PBS or 500 ng of overlapping Min peptides in 0.2 M carbonate/bicarbonate buffer at pH = 9.6 and incubated overnight at 4 °C. After coating, the plate was washed with 1× PBS + 0.05% Tween20 and blocked with 1% BSA + 0.05% Tween20. Then, samples were incubated overnight. Increasing concentrations of mouse monoclonal anti-HIV-1 p24 antibody (clone 39/5.4 A; Abcam) or an anti-gp41 antibody (clone D50, NIH HIV Reagent Program) were used to generate standard curves for Gag and Min, respectively. Results were expressed as p24 or D50 equivalents16 (link). Total IgG was determined with a secondary HRP-conjugated AffiniPure Goat anti-mouse IgG (115-036-071; Jackson ImmunoResearch) at a 1:10,000 dilution. IgG subclasses were determined using Biotin-conjugated AffiniPure Goat anti-mouse IgG1, IgG2b, IgG2c, and IgG3 antibodies (1106205, 115-065-207, 115-065-208 and 115-065-209, respectively; Jackson ImmunoResearch) at a 1:5,000 dilution and an HRP-conjugated Streptavidin (N100; ThermoFisher Scientific) at a 1:6000 dilution. Finally, after five washes, the assay was developed using o-phenylenediamine (OPD; ThermoFisher Scientific) for 10 min and stopped using 4 N H2SO4.
5
Western Blotting Procedure for Protein Analysis
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Western blotting analysis was carried out as detailed elsewhere (60 (link),62 (link)). Fish were sacrificed after anesthesia and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty micrograms of total proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)]. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was carried out as detailed previously (60 (link)).
6
Immunohistochemical Analysis of Mouse Embryonic Development
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For immunohistochemical analysis, embryos at E9.5 were embedded in gelatin (15% sucrose and 7.5% gelatin in PBS) and cryo-sectioned at thicknesses of 10 μm and 50 μm on a Leica CM1950 cryostat. Sections of at least three different embryos per primary antibody were blocked with PBT (PBS, 10% Triton and 1% BSA) and incubated overnight at 4°C with the primary antibody against myosin heavy chain II-B (PRB-445P, Covance) diluted 1:150 in PBT; β-catenin (610153, BD Biosciences) diluted in PBT 1:200; ZO1 (40-2200, Invitrogen) diluted 1:100 in PBT; or Vangl2 (a kind gift from Mireille Montcouquiol, Neurocentre Magendie, Bordeaux, France) (Belotti et al., 2012 (link)) diluted 0.5:150 in PBS and 5% BSA; or Pax6 [mouse monoclonal, Developmental Studies Hybridoma Bank (DSHB)] diluted in PBT 1:200. The secondary antibodies used were as follows: goat anti-rabbit FITC-conjugated for the primary antibodies against myosin II, ZO1 and Vangl2 (ab6717, Abcam) or a Cy™3-conjugated affiniPure goat anti-mouse IgG to detect antibodies staining β-catenin and Pax6 (115-165-166, Jackson ImmunoResearch). Phalloidin-Tetramethylrhodamine B isothiocyanate was used to visualize F-actin (P1951, Sigma-Aldrich). Sections were mounted with Hydromount (HS-106, National Diagnostics), and then photographed by using an Olympus (Tokyo, Japan) BX61 microscope or a confocal microscope (TCS-SP2-AOBS, Leica).
7
Quantifying Collagen I in Right Ventricular Tissue
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Right ventricular tissue, fixed in 4% paraformaldehyde for 24 h, was processed, embedded in paraffin, and subsequently cut into 4-μm-thick sections. The slides then underwent heat-mediated antigen retrieval followed by incubation with primary antibody anti-Collagen 1 antibody-3G3 (Abcam ab88147) and secondary antibody AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Lab Inc, Code 115005003). The slides were then analyzed using an Olympus FluoView FV300 Confocal Laser Scanning Microscope (Thermo Fisher Scientific), and the Fiji image processing software (an open-source platform for biological image analysis) was used for analysis of pixel density (23 (link)).
8
Western Blot Analysis of p53 in Irradiated Cells
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24 hours post irradiation, cells were washed once with PBS and scraped and harvested in sample buffer (2.5% w/v SDS, 25% v/v Glycerol, 125mM Tris pH-6.8, 0.01% w/v bromophenol blue, 4% β-mercaptoethanol, in water). Samples were sonicated for ten seconds (30% amplitude, Branson Digital Sonifier) and heated at 95°C for 5 minutes. Proteins were resolved on 10% SDS-polyacrilamide gels and transferred to 0.45μm nitrocellulose membranes (Bio-Rad). Western blots were probed with anti-mouse p53 (Cell signaling) and GAPDH (Millipore). Antibodies were detected using peroxidase-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Labs) and enhanced chemiluminescence western blotting detection reagents (Thermo Fisher scientific). Gels were Imaged by CemiDoc XRS+ (Bio-Rad) and analyzed by ImageLab 5.1 software (BioRad).
9
Immunostaining for Autophagy Markers
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Mouse anti-DRD2 antibody (1:100; Santa Cruz) was used for the identification of the DRD2-containing neurons. FITC-conjugated AffiniPure goat anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, West Grove, USA) and rhodamin-conjugated AffiniPure goat anti-mouse IgG (1:200, Jackson) were used to recognize rabbit anti-LC3B antibody (1:50; Sigma) and mouse anti- beclin-1(BECN1) antibody (1:50; BD), respectively. DAPI (Sigma) was used for nuclear labeling. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) solution containing FITC-dUTP (Roche, Basel Schweiz, Switzerland) was used for the detection of DNA fragmentation. Autophagic cell death was identified by the presence of TUNEL (+) BECN1 immunoreactive cells with intact nuclei [23 (link)]. The fields selected for quantification are around the lesion site resided in striatum as shown in panel (C) of S2 Fig S2 Fig
10
Quantitative Detection of West Nile Virus
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WNV RNA purification was performed with QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers' instructions. The quantitative real-time PCR was performed using QuantiTec Probe RT-PCR MasterMix Kit (Qiagen, Hilden, Germany), with Light Cycler 480 II (Roche, Rotkreuz, Switzerland) and using 9 μl reaction mix containing 0.6 μM of the following primers and 0.2 μM of the following probes: OSM_145: GGCAATGGAGTCATAATG; OSM_146: GCATCTCAGGTTCGAATC; OSM_147: -FAM-CCAACGGCTCATACATAAGCG-BHQ1 and 2 μl RNA. For the analysis of virus titres, mosquito organ homogenate was filtered using 0.20 μm filters and inoculated on Vero cells (96-well format) with 10-fold dilutions and indirect immunofluorescent revelation after 3 days. Briefly, inoculated Vero cells were fixed in 4% formaldehyde for 30 min and immunostained using WNV recombinant E protein mouse monoclonal antibody (ABIN782271, antibodies-online GmbH, Germany) diluted 1:100 in PBT for 1–2 h and then with fluorescein (FITC)-conjugated AffiniPure goat anti-mouse IgG (115-095-003, Jackson ImmunoResearch Laboratories, Inc., USA) diluted 1:200 in PBT for 1 h. Infected wells were counted and viral titres were calculated using the Spearman and Kärber algorithm described by Hierholzer and Killington [33] .
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