The liver tissues were embedded in OCT and stored at -80°C. OCT-embedded liver tissues were cut into 6μm sections. The sections were fixed in 4% paraformaldehyde for 15 min and then washed with PBS. Next, the sections were incubated in permeabilization solution (0.3% Triton X-100 in PBS) for 15 min and then washed with PBS. Then, the sections were incubated with anti-collagen I (ab34710, Abcam), anti-Ki67 (ab15580, Abcam) or α-SMA (ab7817, Abcam) antibodies. After washing, the sections were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (711-545-152, Jackson ImmunoResearch Labs), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (ab150074, Abcam) or Alexa Fluor 594-conjugated donkey anti-mouse IgG (715-585-150, Jackson ImmunoResearch Labs). The sections were washed with PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI) (10236276001, Roche) and mounted with a cover glass. The images were captured with a confocal laser microscope setup (LSM980, Zeiss) and processed using ZEN (Zeiss).
Alexa fluor 594 conjugated donkey anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama
Alexa Fluor 594-conjugated donkey anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) molecules. The antibody is conjugated with the Alexa Fluor 594 fluorescent dye, which emits red fluorescence when excited by the appropriate wavelength of light. This product can be used for various applications that require the detection of mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Peroxidase linked anti mouse immunoglobulin g, by GE Healthcare (2 mentions)
Peroxidase linked anti rabbit immunoglobulin g, by GE Healthcare (2 mentions)
Ab15580, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab150074, by Abcam (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Vectashield h 1500, by Vector Laboratories (1 mentions)
Tyr701, by Cell Signaling Technology (1 mentions)
488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Bz x800 fluorescence microscope, by Keyence (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Anti actin, by Merck Group (1 mentions)
8 protocols using alexa fluor 594 conjugated donkey anti mouse igg
1
Multimodal Imaging of Liver Fibrosis
2
Imaging of HLA-ABC and Phospho-STAT1
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Cells were incubated on a glass chamber slide with the indicated drug, covered, and incubated with ice-cold 1:1 methanol and acetone mixture for 10 min at −20°C. After washing with PBS, cells were blocked with diluted donkey serum for 30 min at room temperature, then incubated with mouse-anti HLA-ABC antibody (#ab70328, Abcam) and rabbit-anti phosphorylated STAT1 (Tyr701, #7649, Cell Signaling Technology) overnight at 4°C. Cells were washed with PBS and incubated with Alexa Fluor 594-conjugated donkey anti-mouse IgG (#715-585-150, Jackson ImmunoResearch) and 488-conjugated donkey anti-rabbit IgG (#11-545-152, Jackson ImmunoResearch) antibodies, respectively, for 1 hour at room temperature. After washing, slides were mounted with medium containing DAPI (Vectashield H-1500, Vector Laboratories). A BZ-X800 fluorescence microscope (Keyence Corporation) was used to assess the expression and subcellular localization of HLA-ABC and phosphorylated STAT1.
3
Immunofluorescence Staining of RPE Cells
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After fixation with 4% paraformaldehyde in PBS for 15 min, cells were permeabilized using 0.25% Triton X-100 in PBS for 5–10 min and were blocked for 60 min in 5% goat serum. Then, the cells were incubated for 60 min at room temperature with the following primary antibodies: anti-MITF antibody (Sangon, Cat# D120988, 1:100), anti-ZO-1 antibody (Thermo Fisher Scientific Cat# 40-2200, RRID:AB_2533456 , 1:200), anti-Bestrophin antibody (Novus Cat# NB300-164, RRID:AB_10003019 , 1:100), anti-RPE65 antibody (Abcam, Cat# ab78036, RRID:AB_1566691 , 1:100), anti-PMEL-17 antibody (Abcam, Cat# ab137078, RRID:AB_2732921 , 1:100), Cells were then incubated for 120 min at room temperature with the corresponding secondary antibodies: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat# 711-586-152, RRID:AB_2340622 ), Alexa Fluor 594-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat# 715-586-150, RRID:AB_2340857 ), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat# 711-546-152, RRID:AB_2340619 ) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat# 715-545-150, RRID:AB_2340846 ). Fluorescence images were acquired with a confocal microscope (Zeiss LSM 800, Carl Zeiss).
4
UNC-45A Protein Detection Protocols
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Rabbit anti-UNC-45A raised against the C-terminus of the human UNC-45A (Protein Tech 1956–1-AP) was used for Western blot and immunofluorescence at the concentration recommended by the manufacturer (1:1000 dilution for Western blot and 1:200 dilution for immunofluorescence) and previously used in our laboratory5 . Mouse polyclonal anti-UNC-45A raised against full-length UNC-45A (Abnova) was used for Western blot at the concentration recommended by the manufacturer (1:300 dilution) and as previously described.6 Anti-NMII (LC) (Sigma), anti-actin (Sigma), and anti-alpha-tubulin (Abcam) were used at the concentration recommended by the manufacturers. Peroxidase-linked anti-mouse immunoglobulin G and peroxidase-linked anti-rabbit immunoglobulin G (both GE Healthcare Bio-Sciences, Pittsburgh, PA). Alexa Fluor 594-conjugated Donkey Anti-Mouse IgG (1:250) and FITC-conjugated Goat Anti-Rabbit IgG (1:200; both Jackson ImmunoResearch Laboratories, West Grove, PA). Texas-Red conjugated Goat Anti-Mouse IgG (1:250; both Jackson ImmunoResearch Laboratories, West Grove, PA), and FITC-conjugated Goat Anti-Mouse IgG (1:250; both Jackson ImmunoResearch Laboratories, West Grove, PA).
5
Immunofluorescence Staining of PRRSV-N Protein
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Cells seeded in 24-well plates were fixed with 4% paraformaldehyde (precooled at 4 °C) for 10 min and then immediately permeabilized with methanol (precooled at −30 °C) for 15 min prior to the addition of blocking reagent (5% BSA). Next, cells were incubated with the primary antibody against PRRSV-N protein for 1 h and washed three times with PBS, followed by incubation with Alexa Fluor 594-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, PA, USA). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) for 45 min. Fluorescent images were acquired with Axio-Imager_LSM-800 (Zeiss, Germany).
6
Fluorescent Immunohistochemistry of Tissue Sections
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Fluorescent immunohistochemistry was performed on cryosectioned tissue as previously described51 (link). Antibodies utilized for fluorescent immunohistochemistry are as follows: goat anti-GFP (1:500; Rockland Immunochemicals, Limerick, PA), rabbit anti-GFP (1:1000; Invitrogen, Waltham, MA), mouse anti-Glutamine synthase (GLUL) (1:200; BD Biosciences, San Jose, CA), mouse anti-KI67 (1:200; BD Biosciences, San Jose, CA), rabbit anti-LHX2 (1:1500; generated in house with Covance, Princeton, NJ), mouse anti-NFIA (1:200; CDI Laboratories), mouse anti-P27Kip1 (1:200; Invitrogen), mouse anti-PAX6 (1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-phosphohistone H3 (PHH3) (1:200; Millipore, Billerica, MA). Secondary antibodies used were as follows: AlexaFluor488 conjugated donkey anti-goat IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor488 conjugated donkey anti-rabbit IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor594 conjugated donkey anti-rabbit IgG (1:500; Jackson Immunoresearch, West Grove, PA), AlexaFluor594 conjugated donkey anti-mouse IgG (1:500; Jackson Immunoresearch, West Grove, PA). All section immunohistochemical data shown was imaged and photographed on a Zeiss Meta 510 LSM confocal microscope.
7
Comprehensive characterization of extracellular vesicles
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The concentration of EVs was estimated based on the total protein content, determined using a bicinchoninic acid (BCA) protein assay kit (Cat. No. E-BC-K318-M, Elabscience). Nanoparticle tracking analysis (NTA) with a ZetaView PMX 110 (Particle Metrix) was used to analyze the distribution and size of EVs, while Hitachi H-7650 transmission electron microscope (TEM; Hitachi) was used to identify the morphologies of EVs. To assess the effectiveness and stability of each batch of EVs, cellular functional tests and NTA analysis were employed. Flow cytometry on a FACSCANTO II (BD Biosciences) was employed to resolve EV surface marker proteins, including CD63, CD81, and TSG101, as previously described [48 (link), 49 (link)], and FlowJo software (Tree Star Inc) was used to analyze the results. The CD63 polyclonal antibody (Cat. No. 25682-1-AP, Proteintech), CD81 monoclonal antibody (Cat. No. SC7637, Santa Cruz Biotechnology), TSG101 polyclonal antibody (Cat. No. 14497-1-AP, Proteintech) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rabbit IgG (Cat. No. 111-545-144, Jackson ImmunoResearch) and Alexa Fluor 594-conjugated donkey anti-mouse IgG (Cat. No. 715-585-151, Jackson ImmunoResearch) were used as secondary antibodies.
8
Detecting UNC-45A in Cells
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Rabbit anti UNC-45A (Protein Tech, 1956-1-AP) raised against the C-terminus of the human UNC-45A (Protein Tech, 1956-1-AP) was used for Western blot and immunofluorescence as we have previously described (Habicht et al., 2019a; Mooneyham et al., 2019) . Mouse polyclonal anti UNC-45A raised against full length UNC-45A (Abnova, H00055898-B01P) was used for Western blot to detect recombinant UNC-45A and its mutants (Figure 3B). Mouse anti-αtubulin (Sigma-Aldrich, T6074), for Western blot, rabbit polyclonal α -tubulin (Abcam, ab18251), mouse monoclonal anti-detyrosinated-α-tubulin (EMD Millipore AB3201), rabbit monoclonal anti-GFP (Thermo Fisher Scientific G10362), mouse monoclonal anti-flag (Sigma-Aldrich, F1804), Peroxidase-linked anti-mouse immunoglobulin G and peroxidase-linked antirabbit immunoglobulin G (both Cytiva, formerly known as GE Healthcare Bio-Sciences NA931 and NA934), Alexa Fluor 594-conjugated Donkey Anti-Mouse IgG (1:250) and FITCconjugated Goat Anti-Rabbit IgG (1:200; both Jackson ImmunoResearch Laboratories 715-585-150 and 111-095-003). Paclitaxel was purchased from Teva Pharmaceuticals. Blebbistatin was purchased from Sigma-Aldrich, St. Louis, MO. Tubulin Tracker Deep Red was purchased from Thermo Fisher Scientific (Waltham, MA) cat # T34077.
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