Flp-In TREx293 Gpr176-GFP (tet-on) cells and the related cells that express an N-terminal N-glycosylation mutant Gpr176-GFP were plated on polylysine-coated coverslips in culture medium containing 1 µg/ml Dox for 15 h. The cells were fixed with 4% paraformaldehyde and mounted in ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific). The Gpr176 GFP fusion protein subcellular localization was monitored using direct fluorescence from the GFP moiety. For ER staining, cells were treated with 1 μM ER-Tracker Red (Thermo Fisher Scientific). ER-Tracker is a fluorescence-labeled glibenclamide that is capable of visualizing ER via specific binding to the sulphonylurea receptors of ATP-sensitive K+ channels, which are prominent on ER40 (link). For calnexin immunocytochemistry, cells were fixed, permeabilized, and blocked with 5% bovine serum albumin in PBS containing 0.1% Triton X-100, as described41 (link). The cells were immunolabeled with anti-calnexin (BD Transduction Laboratories, #610523) and visualized with Alexa594-conjugated anti-mouse IgG (Thermo Fisher Scientific). Images were captured using an Olympus FV10I-DOC confocal microscope.
Alexa594 conjugated anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is labeled with the Alexa Fluor 594 fluorescent dye. This antibody can be used for the detection and visualization of mouse IgG in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Vectashield, by Vector Laboratories (4 mentions)
Confocal fluorescence microscope, by Olympus (3 mentions)
Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Mab1572, by Merck Group (2 mentions)
Er tracker red, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
S5768, by Merck Group (1 mentions)
Mab377, by Merck Group (1 mentions)
Prolong antifade kit, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti goat secondary antibody, by Jackson ImmunoResearch (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Fv1000 d ix81 confocal laser scanning microscope, by Olympus (1 mentions)
Anti tiar, by BD (1 mentions)
Fv10 asw software, by Olympus (1 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Bz7000 microscope, by Keyence (1 mentions)
Fluoview 500 9 71 confocal microscope, by Olympus (1 mentions)
14 protocols using alexa594 conjugated anti mouse igg
1
Visualizing GPR176 Protein Localization
2
Immunoreactivity of Aβ Accumulation and Neuronal Markers in Retina and Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the immunoreactivity of Aβ accumulation, the retina and brain tissues were incubated using a blocking solution (0.05% bovine serum albumin, 1.5% normal goat serum, and 0.3% Triton X-100 in PBS) and mouse anti-4G8 antibody specific for Aβ17-24 (1:2000; BioLegend, San Diego, CA, USA) overnight at 4 °C. To examine the immunoreactivity of neuronal nuclei (NeuN) and synaptophysin (SYN), free-floating brain sections were incubated overnight at 4 °C with the mouse anti-NeuN antibody (1:1000; Cat.# MAB377, Merk KGaA, Darmstadt, Germany) or mouse anti-SYN antibody (1:500; Cat.#S5768, Sigma-Aldrich), respectively, in blocking solution. Additionally, the retina and brain tissues were incubated with donkey Alexa 488-conjugated anti-mouse IgG (1:200; Thermo Fisher Scientific Inc., Waltham, MA, USA) or donkey Alexa 594-conjugated anti-mouse IgG (1:200; Thermo Fisher Scientific Inc.) for 1 h at room temperature. To counterstain the nuclei, the retina and brain tissues were mounted on ProbeOnTM Plus Microscope Slides (Thermo Fisher Scientific Inc.) and covered with FluoroshieldTM with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).
3
Immunohistochemical Characterization of Cryab Expression in Rat Medial Prefrontal Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the immunohistochemistry, mother and pup-deprived rats (3-3) were deeply anesthetized and fixed with 4% paraformaldehyde via transcardially perfusion. Brains were removed and postfixed in 4% paraformaldehyde for 24 h then transferred to 20% sucrose in phosphate buffer (PB, pH=7.4) for 48 h. Serial coronal brain sections were cut at 40 µm on a sliding microtome. Sections were collected in PB containing 0.05% sodium-azide and stored at 4 o C. The mPFC sections were incubated in anti-Cryab primary antibody (1:20 dilution) followed by a biotinylated anti-goat secondary antibody (1:1,000 dilution, Jackson Immunoresearch, West Grove, PA). Visualization was performed using an ABC kit (1:500 dilution, Vector Laboratories, Burlingame, CA, USA) and a Ni-DAB reaction. For double labeling, the visualization of Cryab was performed using FITC-tyramide (1:8,000)
A C C E P T E D M A N U S C R I P T amplification followed by incubation with mouse anti-calbindin (1:900 dilution, catalogue number: C9848, Sigma) or anti-parvalbumin (1:800 dilution, catalogue number: P3088, Sigma) antibodies, which were visualized with Alexa 594-conjugated anti-mouse IgG (1:400 dilution, Thermo Fisher Scientific, Waltham, MA). Subsequently, the sections were mounted, dried, and coverslipped in antifade medium (Prolong Antifade Kit; Molecular Probes).
A C C E P T E D M A N U S C R I P T amplification followed by incubation with mouse anti-calbindin (1:900 dilution, catalogue number: C9848, Sigma) or anti-parvalbumin (1:800 dilution, catalogue number: P3088, Sigma) antibodies, which were visualized with Alexa 594-conjugated anti-mouse IgG (1:400 dilution, Thermo Fisher Scientific, Waltham, MA). Subsequently, the sections were mounted, dried, and coverslipped in antifade medium (Prolong Antifade Kit; Molecular Probes).
4
Visualizing Stress Granule Dynamics in COS-7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 24 h of transfection with DsRed-monomer-DGKη1 and/or EGFP-ASK3, COS-7 cells were incubated in DMEM with or without 500 mM sorbitol for 30 min. The cells were fixed in 3.7% paraformaldehyde. The coverslips were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence imaging was performed using an Olympus FV1000-D (IX81) confocal laser scanning microscope (Olympus, Tokyo, Japan). Images were acquired using FV-10 ASW software (Olympus).
To observe stress granule markers, Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and T-cell intracellular antigen 1 related protein (TIAR), cells were fixed and then permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 and 1% bovine serum albumin. Coverslips were incubated with an anti-G3BP1 (Cat. #: 611126, BD Biosciences, Franklin Lakes, NJ, USA) or anti-TIAR (Cat. #: 610352, BD Biosciences) mouse monoclonal antibody for 1 h and then incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probe) for 1 h.
To observe stress granule markers, Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and T-cell intracellular antigen 1 related protein (TIAR), cells were fixed and then permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 and 1% bovine serum albumin. Coverslips were incubated with an anti-G3BP1 (Cat. #: 611126, BD Biosciences, Franklin Lakes, NJ, USA) or anti-TIAR (Cat. #: 610352, BD Biosciences) mouse monoclonal antibody for 1 h and then incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probe) for 1 h.
5
Immunofluorescence Staining of Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure was essentially as described previously [37 (link)]. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, incubated with a rabbit polyclonal anti-MAP2 antibody (1:200; Millipore Japan, Osaka, Japan) and a mouse monoclonal anti-phospho-neurofilament (pNF) antibody (1:250; Covance Japan, Tokyo, Japan), and with species-specific fluorophore-conjugated secondary antibodies (1:1000; Alexa 488-conjugated anti-rabbit IgG and Alexa 594-conjugated anti-mouse IgG; Molecular Probes, Tokyo, Japan). Fluorescent images were captured using a BIO-REVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).
6
Fluorescent Imaging of Hyaluronan and CD44 in Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spheroids were fixed with cold 4% PFA in PBS. After blocking with Block Ace (DS Pharma Biomedical), the spheroids were permeabilized with 0.1% saponin and stained with FITC-conjugated anti-CD44 mAb (clone; IM7, BioLegend) plus biotin-conjugated hyaluronan-binding protein (HABP) (Seikagaku Corp.) After a PBS wash, the spheroids were stained with Alexa594-conjugated streptavidin and 4′, 6-diamidino-2-phenylindole (DAPI). For the analyses of Ki-67 expression, anti-Ki-67 mAb (Becton Dickinson) and Alexa594-conjugated anti-mouse IgG (Molecular Probes) were used. In some experiments, spheroids were treated with hyaluronidase (100 U/ml; Sigma) and incubated with FITC-labeled HA (FL-HA; Seikagaku Corp.) in the presence or absence of anti-CD44 mAb (clone; BRIC 235). Fluorescent images were taken with the BZ-7000 microscope (Keyence). The specificity of the HABP was confirmed by hyaluronidase treatment before HABP staining.
7
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was carried out as described previously [26] .
Alexa-488-conjugated anti-rabbit IgG or Alexa-594-conjugated anti-mouse IgG (Molecular Probes) were used to visualize the localization of the target proteins.
Alexa-488-conjugated anti-rabbit IgG or Alexa-594-conjugated anti-mouse IgG (Molecular Probes) were used to visualize the localization of the target proteins.
8
Immunohistochemical Analysis of Parvalbumin-Positive Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were deeply anaesthetized with fatal plus (pentobarbital) and perfused with saline followed by 4 %paraformaldehyde in 0.1M phosphate buffer. The brain was removed and post-fixed for 24h at room temperature. After fixation, the brain was sectioned into 60 μm coronal slices using a vibratome. Slices were incubated with blocking solution (10 % fetal bovine serum in PBS with 0.2 % Triton X-100) for 1 hour at room temperature and then with anti-Parvalbumin mouse primary antibody (MAB1572, Millipore; 1:1,000) diluted in blocking solution overnight at 4 degrees Celsius. Slices were then washed three times with PBS and incubated with the secondary antibody for 1h at room temperature (Alexa594-conjugated anti-mouse IgG, Invitrogen, 1:500. Slices were washed three times with PBS and mounted with 49,6-diamidino-2 phenylindole (DAPI)-containing Vectashield (Vector Laboratories). Fluorescence images were taken with a confocal fluorescence microscope (Olympus).
9
Immunofluorescence Analysis of FXR and β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 30 minutes, washed 3 times with PBS, and permeabilized by incubation in PBS with 0.1% Triton X-100 for 10 minutes. After blocking in PBS with 10% goat serum and 0.1% Triton X-100 for 1 hour at room temperature, cells were incubated with the primary antibodies (anti-FXR, Abmart, China) monoclonal mouse antibody and anti-β-Catenin rabbit antibody (Cell Signaling) at 4°C overnight, followed by incubation with the secondary antibodies (Alexa488-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) and Alexa 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) at room temperature for 2 hours. Coverslips were mounted onto glass slides with anti-fade mounting medium with DAPI (Vector Laboratories) and visualized with an Olympus FluoView 500 IX71 confocal microscope.
10
In vitro myelination assay for quantifying myelin profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro myelination assays were performed as described previously15 (link). In some experiments, myelination was induced with mGluRs antagonist at concentrations mentioned above. For analysis of myelin profiles by immunocytochemistry, 10 days after initiation of myelination, the culture was fixed with 4% paraformaldehyde/PBS for 5 min. Myelination profiles were visualized by immunocytochemical detection of myelin basic protein (MBP) using anti-MBP antibody (Covance) followed by Alexa-594-conjugated anti-mouse IgG (Invitrogen). For quantification, myelination profiles in 5 randomly chosen fields using a 20x objective lens were counted and the number of myelinated nerve fibers per arbitrary unit area was calculated. Statistical analysis was performed by Student’s t test. All data are reported as mean ± SD; p value of less than 0.05 is considered significant.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!