KU-63794 was obtained from CalBiochem and AZD8055 was purchased from Selleckchem. Phycoerythrin (PE) Cy7-conjugated anti-Sca-1 (Clone E13-161.7, rat IgG2a), APC-conjugated anti-c-kit (Clone 2B8, rat IgG2b), and purified rat anti-CD 16/CD32 (Clone 2.4G2, Fcγ receptor blocker, rat IgG2b) were purchased from BD Pharmingen (San Diego, CA). Both mouse and human Hematopoietic Progenitor (Stem) Cell Enrichment Set-DM were purchased from BD Biosciences. Recombinant mouse thrombopoietin (TPO) was purchased from R&D Systems (Minneapolis, MN). The rabbit anti-phospho-p70 S6 kinase (p70S6K) monoclonal antibody and active (cleaved) caspase-3 antibodies were purchased from Cell Signaling. The Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody was purchased from Invitrogen (Carlsbad, CA).
Alexa fluor 594 conjugated goat anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody is a secondary antibody that binds to rabbit immunoglobulin G (IgG) molecules. The antibody is conjugated with the Alexa Fluor 594 fluorescent dye, which enables detection and visualization of target proteins in various applications, such as immunofluorescence microscopy and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Cs sp5, by Leica (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Rat igg2b, by BD (1 mentions)
Active cleaved caspase 3 antibodies, by Cell Signaling Technology (1 mentions)
Recombinant mouse thrombopoietin tpo, by R&D Systems (1 mentions)
Azd8055, by Selleck Chemicals (1 mentions)
Tcs sp5 laser confocal microscopy, by Leica (1 mentions)
Slowfade reagent, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse monoclonal anti myc antibody, by Cell Signaling Technology (1 mentions)
Hoechst, by Vector Laboratories (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
A0452, by Agilent Technologies (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Dapi mounting medium h 1500, by Vector Laboratories (1 mentions)
X0909, by Agilent Technologies (1 mentions)
17 protocols using alexa fluor 594 conjugated goat anti rabbit igg antibody
1
Hematopoietic Stem Cell Enrichment and Analysis
2
Immunohistochemical Staining of Ileal Tissue
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Frozen sections of ileal tissue were fixed with 4% paraformaldehyde for 10 min at room temperature, then washed thrice with PBS, permeabilized in 0.1% Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, the nonspecific binding sites were blocked with 5% normal goat serum in PBS for 30 min. Then, the sections were incubated with monoclonal rabbit antibody against ZO-1 (1:200, Invitrogen), ASC (1:100, Cell Signaling), LC3B (1:100, Sigma) at 4°C overnight. After washing thrice in PBS, sections were incubated with secondary Alexa Fluor 594 conjugated goat anti-rabbit IgG antibody (1:200, Invitrogen) and DAPI (1:1000, Sigma) for 1 hour at room temperature. After washing thrice in PBS, sections were mounted using Slowfade reagents (Invitrogen). Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).
3
Immunostaining of Cultured Parasite Smears
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Thin blood smears from cultured parasites were prepared, air-dried and fixed in a 1:1 acetone:methanol mixture at − 30 °C for 5 min31 (link). Smears were blocked with PBS containing 10% normal goat serum (Invitrogen) at 37 °C for 30 min and immunostained with mouse anti-myc monoclonal antibody (9B11, Cell Signaling Technology) at 1:500 dilution in PBS supplemented with 0.05% Tween-20 and incubated at 4 °C overnight. Double immunostaining of smears was done with rabbit anti-SBP4 at 1:1000 dilution. The smears were incubated with Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen) at 37 °C for 30 min. Nuclei were stained by incubation of smears with 1 μg/mL Hoechst 33342 solution. The smears were examined using a confocal laser-scanning microscope (CS-SP5, Leica Micro-system, Wetzlar, Germany).
4
Quantification of Neutrophil Extracellular Traps
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After 2 h of incubation, the medium containing the reagents was removed, and the remaining cells were washed with PBS followed by fixation with 4% paraformaldehyde for 15 min. Thereafter, the specimens were made to react with 5 μg/ml of rabbit anti-human citrullinated histone 3 polyclonal antibody (Abcam, Cambridge, UK) for 60 min at room temperature. After removal of unbound antibody, the specimens were next allowed to react with 1:500 dilution of Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (Invitrogen, Tokyo, Japan) for 60 min at room temperature. After washing with PBS, the specimens were finally mounted with the 4′, 6-diamidino-2-phenylindole (DAPI)-containing solution (Sigma-Aldrich). NET formation was observed under a fluorescent microscope and was quantified by counting the citrullinated histone 3-positive cells per × 100 power field of view. Data from five random fields of view (×100) were subjected to the quantitative analysis.
5
Immunocytochemical Analysis of Wnt7a and α-SMA
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For the immunocytochemical analysis, cells were fixed in 4% paraformaldehyde for 30 min at 4°C and then permeabilized by 0.1% Triton X-100 for 15 min, followed by blocking with 3% fetal bovine serum for 30 min. Primary antibodies: rabbit polyclonal antibodies to Wnt7a and α-SMA (Abcam, MA, UK) were incubated with the cells overnight at 4°C. Secondary antibodies: an Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody and an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen, CA, USA) were added to the cells and incubated for 1 h at 37°C. Finally, the cells were stained with Hoechst (Vector, Burlingame, CA) at a 1 : 3000 dilution and were examined under a fluorescence microscope (Olympus BX53, JP).
6
Immunofluorescence Staining of FFPE Slides
7
Confocal Microscopy of HCAEC-PBMC Interaction
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HCAECs were seeded in confocal microscopy plates (diameter: 35 mm; glass bottom diameter: 20 mm) at a density of 5×103 cells per well and cultivated as described above. Meanwhile, PBMCs were grown in the upper chamber of the Transwell insert (PET 0.4 µm, available for 6-well plate). After 48 h of co-cultivation with PBMCs, HCAECs were immediately fixed with cold 4% paraformaldehyde for 30 min, washed with PBS (5 min ×3), permeabilized with 0.5% Triton X-100, and blocked with 1% bovine serum albumin (BSA) for 30 min. Subsequently, the cells were incubated overnight with anti-LC3 II/I antibody (1:200; Cat: 4108; CST, USA) at a dilution of 200 times in 0.5% BSA. After three times of gentle washing with PBS, the cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:200; Cat: A-11012; Invitrogen, Carlsbad, CA, USA). Nuclei were identified by DAPI staining. Olympus FV1000 (IX81) confocal microscope (Tokyo, Japan) was used to capture the fluorescent images.
8
Immunostaining of Cultured Parasites
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Thin blood smears from cultured parasites were prepared, air-dried and fixed in a 1:1 acetone:methanol mixture at -30°C for 5 min 29 . Smears were blocked with PBS containing 10% normal goat serum (Invitrogen) at 37°C for 30 min and immunostained with mouse anti-myc monoclonal antibody (9B11, Cell Signaling Technology) at 1:500 dilution in PBS supplemented with 0.05% Tween-20 and incubated at 4°C overnight. Double immunostaining of smears was done with rabbit anti-SBP4 at 1:1000 dilution. The smears were incubated with Alexa fluor ® 488conjugated goat anti-mouse or Alexa fluor ® 594-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen) at 37°C for 30 min. Nuclei were stained by incubation of smears with 1 μg/mL Hoechst 33342 solution. The smears were examined using a confocal laser-scanning microscope (CS-SP5, Leica Micro-system, Wetzlar, Germany).
9
Neutrophil NET Formation Assay
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Neutrophils from a healthy volunteer were seeded in the wells of four-well chamber slides (1 × 10 6 /ml), treated with 5 ng/ml TNF-α for 15 min at 37ºC, and then exposed to 250 μg/ml of the eluted IgG samples from MPA patients (n=19) and healthy individuals (n=8). After incubation for 3 h at 37ºC, the supernatants were removed and then the remaining cells on the slides were fixed with 4% PFA for 15 min at room temperature. Thereafter, the samples were washed with PBS and then allowed to react with rabbit anti-Cit-H3 antibody (1:100) for 1 h at room temperature. After washing with PBS, the samples were next exposed to Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen) for 1 h at room temperature followed by mounting with the DAPI-containing solution. Photomicrographs (magnification, ×200) were taken randomly (six fields per well of chamber slides), and then the number of Cit-H3-positive and DAPI-positive NETs was counted using the ImageJ software (free software; https://imagej.nih.gov/ij/index.html). Data were standardized by the number of DAPI-positive neutrophils.
10
Immunohistochemical Analysis of ARE-luc2 Mouse Brains
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ARE-luc2 mice were sacrificed and perfused transcardially with cold saline solution followed by 4% paraformaldehyde in PBS. Brains were immediately removed, post-fixed in the 4% paraformaldehyde fixative for 24 h and then transferred in solutions of sucrose at increasing concentrations (up to 30%) during the following 72 h. Samples were then frozen and stored at −80°C for successive analyses. Serial coronal sections of 40 μm were cut throughout the brain using a freezing sliding microtome (Leika SM 2000R) and stored at −20°C in a solution containing 30% ethylene glycol, 20% glycerol and 0.05 M sodium phosphate buffer until use. The slide-mounted sections were rinsed in PBS and incubated in PBS containing 10% NGS and 0.3% TX-100 at room temperature. Sections were then incubated at 4°C in PBS/1% NGS/0.3% TX-100 containing a rabbit polyclonal anti-RFP antibody (diluted 1:400; Abcam, Cambridge, UK). After a 24 h incubation sections were rinsed in PBS and incubated 1 h at RT in PBS/1% NGS containing the Alexa Fluor 594 conjugated goat anti-rabbit IgG antibody (1:300; Molecular Probes, Carlsbad, CA, USA). Finally, sections were rinsed in PBS and covered with Prolong with DAPI (Molecular Probes, Carlsbad, CA, USA).
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