Paraffinized heart tissue sections (4 µm in thickness) were steamed for 15 min in the Trilogy solution from Sigma-Aldrich (Cat. No. 920 P) for deparaffinization and antigen retrieval. Afterwards, the slides were blocked with 1% BSA in TBS-T solution followed by incubating overnight at 4 °C with anti-CD31 antibody from Abcam (Cat. No. ab28364, 1:50 dilution) or anti-CD34 antibody from R&D (Cat. No. AF6518). Alexa fluor 594 conjugated anti-rabbit antibody from ThermoFisher) (Cat. No. A-11012, 1;100 dilution) was used to visualize the CD31 immunostaining, and Alexa fluor 594 conjugated anti-sheep antibody from Abcam (Cat. No. ab96937) was used to visualize CD34. For co-immunostaining of CD31 and CD34, the secondary antibody for CD31 was switched to Alexa fluor 488 conjugated anti-rabbit antibody from Abcam (Cat. No. ab150073). Fluorescent images of CD31 and CD34 staining were taken using a Leica confocal microscope. In each slide, 5 images were randomly taken from different areas of the slide. Images were analyzed using FiJi ImageJ software to calculate the percentage of CD31 positive areas.
Alexa fluor 594 conjugated anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated anti-rabbit antibody is a secondary antibody used in various immunodetection techniques. It is specifically designed to detect and bind to rabbit primary antibodies, allowing for visualization and detection of target proteins or antigens in samples.
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Lab products found in correlation
Alexa fluor 488 conjugated anti mouse antibody, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (3 mentions)
Lsm 880, by Zeiss (2 mentions)
Fluorescence microscope, by Zeiss (2 mentions)
Phospho histone γ h2ax antibody ser139, by Merck Group (2 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (2 mentions)
μ slide 8 well, by Ibidi (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Confocal microscope, by Leica (1 mentions)
Trilogy solution, by Merck Group (1 mentions)
Anti cd34 antibody, by R&D Systems (1 mentions)
Alexa fluor 488 conjugated anti rabbit antibody, by Abcam (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Ab2175, by Abcam (1 mentions)
Anti ha antibody, by BioLegend (1 mentions)
Streptavidin alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Dm6000, by Leica (1 mentions)
Tcs sp8 sted, by Leica (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Las x, by Leica (1 mentions)
19 protocols using alexa fluor 594 conjugated anti rabbit antibody
1
CD31 and CD34 Immunostaining of Heart Tissue
2
Quantifying DNA Damage Response Markers
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Cells were treated with 25 ng/ml illudin S for 1 h and incubated for the indicated times without the drug. Detergent-soluble materials were eliminated by incubation with 0.5% Triton X-100 in PBS on ice for 5 min. The cells were then fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. The fixed cells were blocked with 3% BSA in PBS for 30 min at room temperature and stained using mouse anti-RPA2 (ab2175; Abcam) and rabbit anti-γH2AX (9718; Cell Signaling Technology). RPA and γH2AX signals were visualized with Alexa Fluor 488–conjugated anti-mouse antibody (Thermo Fisher Scientific) and Alexa Fluor 594-conjugated anti-rabbit antibody (Thermo Fisher Scientific), respectively. Nuclei were stained with 2 μg/ml Hoechst 33342. Images were collected using an LSM710 confocal microscope with Plan-Apochromat 40×/1.3 NA Oil (Zeiss). Fluorescence intensities were quantified using Zeiss Zen software.
3
Visualizing Protein Biotinylation in HeLa Cells
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HeLa cells (American Type Culture Collection, ATCC, CCL-2) were transfected with vectors containing MAC-tagged gene of interest and cultured either with or without supplemental biotin. Bait proteins were detected with anti-HA antibody (Biolegend, MMS-101R, dilution 1:500), followed by Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, A-11001, 1:800). Biotinylated proteins were detected with Alexa Fluor 594 streptavidin (Thermo Fisher Scientific; S11227, 1:800). DAPI staining was used to determine the nuclei. Selected endogenous proteins were detected with specific antibodies (Supplementary Data 1a ) and subsequently with Alexa Fluor 594 -conjugated anti-rabbit antibody (Thermo Fisher Scientific, A11012, dilution 1:800). Wide-field fluoresce microscope (Leica, Leica DM6000, Welzlar, Germany) with HCXPL APO 63×/1.40–0.60 oil objective was used to image the samples. For imaging sub-mitochondrial proteins, confocal microscopy (Leica TCS SP8 STED, Leica) with HC PL APO 93×/1.30 motCORR glycerol object was used. The image files were processed with LAS X (Leica), and ImageJ softwares.
4
Immunofluorescence Staining of Cells and Organoids
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Immunofluorescence staining was performed as previously described 38 (link). Briefly, cells grown on coverslips or the sectioned organoids (7 μm) were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100. Following blocking with 10% goat serum, the samples were incubated with the indicated primary antibodies in blocking buffer (10% goat serum in PBS) overnight at 4 °C, and then rinsed and incubated with secondary Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 594-conjugated anti-rabbit antibody (Life Technologies) for 1 h at room temperature. Cells were then rinsed with PBS, stained with 4,6-diamidino-2-phenylindole (DAPI) and mounted. The slides were observed with a fluorescence microscope (LSM880, ZEISS) at Instrumental Analysis Center of Shenzhen University.
5
Hypoxia-Induced Nrf2 Activation by Nifedipine
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A549 cells were seeded at a density of 1 × 105 cells/coverslip and incubated for 24 h. Further, hypoxia was induced using 600 μM CoCl2 (Sigma Aldrich, Switzerland) and incubated at 37 °C for 24 h. After inducing hypoxia for 24 h, cells were treated with 10 μM Nifedipine (Sigma Aldrich, China). Control cells (without any treatment) and hypoxic cells with 600 μM CoCl2 were also maintained at similar conditions. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH, 7.4) at room temperature for 20 min. The cells were then washed three times with PBS and permeabilized with 0.5% Triton X-100 for 15 min. After that, cells were blocked with 2% BSA (Sigma-Aldrich) diluted in PBS for 2 h at room temperature. The cells were incubated anti-Nrf2 human polyclonal primary antibody (1:300, Santa Cruz Biotechnology, Dallas, TX, USA) for 12 h at 4 °C. After that, cells were washed three times with PBS and then incubated with a secondary antibody (Alexa Fluor 594-conjugated anti-rabbit antibody, 1:600 dilutions, Life technologies) for 2 h at 4 °C. Afterward, the cells were washed two times with PBS and stained with Hoechst for 10 min at room temperature. The images were acquired with a laser scanning confocal microscope fluorescence microscope (Leica TCS SP8, USA).
6
Cellular Localization of Brucella Strains
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HeLa epithelial cells were seeded in 24-well tissue culture plates on 12-mm-diameter glass coverslips 2 days before infection to obtain a final concentration of 5 × 105 cells per well and infected with Ba2308, BaR-, BaR-RD58E, BaR-R, and BaR-RD58A at a MOI of 500 as described above. At 48 h post-infection, coverslips were washed three times in 1X PBS and fixed using 3% paraformaldehyde in PBS (pH 7.4) at 37°C for 10 min. Rabbit anti-calnexin polyclonal antibody ab75801 (Abcam) was used to localize intracellular compartments. As reported elsewhere, in house polyclonal mouse antibodies to B. abortus were used to detect Brucella (Altamirano-Silva et al., 2021 (link)). An Alexa Fluor 488-conjugated goat anti-mouse antibody and an Alexa Fluor 594-conjugated anti-rabbit antibody (Life Technologies) were used as developing antibodies. Confocal analysis was performed with an Olympus U-TB190 (100X) under oil immersion. Confocal images of 1,024 by 1,024 pixels were acquired with the FV10-AV ver.03.01 software (Olympus) and assembled with Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).
7
Immunofluorescent Quantification of Tau Aggregates
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For immunofluorescence, HEK293T cells were washed in PBS and fixed in 4% paraformaldehyde for 10 min. Autofluorescence eliminator reagent (Millipore, Burlington, MA) was added for five minutes and washed with 40% ethanol. Under dark conditions, slides with cells were incubated in 0.0125% Thioflavin S dissolved in 50% ethanol/ PBS for 3 min and washed in 50% ethanol and PBS. Slides were submerged in blocking solution (2% FBS/0.1% Triton-X-100 in PBS) for 30 min. Primary antibody in 2% FBS/PBS was incubated for one hour. After PBS washes, Alexa-fluor 594 conjugated anti-rabbit antibody (Invitrogen, Carlsbad, CA) were added at 1: 500 dilution for one hour. Slides were washed in PBS and placed in 0.5 μg/mg of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) in PBS for 5 min. The coverslips were mounted using Fluoromount-G (Invitrogen, Carlsbad, CA). Fluorescent images were captured with a BZ-X700 Keyence digital microscope (Itasca, Il).
For quantification, different 20X fields of ~20–50 cells were captured for each treatment group using a BZ-X700 Keyence digital microscope (Itasca, Il). Tau positive cells that colocalized with Thioflavin S positive aggregates were calculated as a ratio to total number of tau positive cells and reported as a percentage of Thioflavin S positivity.
For quantification, different 20X fields of ~20–50 cells were captured for each treatment group using a BZ-X700 Keyence digital microscope (Itasca, Il). Tau positive cells that colocalized with Thioflavin S positive aggregates were calculated as a ratio to total number of tau positive cells and reported as a percentage of Thioflavin S positivity.
8
DNA Damage Quantification in Cells
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Cells (1 × 106 cells/well) were seeded on 13-mm glass coverslip in 6-well plates. After treatment, cells were washed and fixed with 1% paraformaldehyde (Sigma-Aldrich) and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. The samples were blocked with 1% BSA for 1h and incubated with phospho-histone-γ-H2AX antibody (Ser139) (Millipore) and p53-binding protein 1 (53BP1) antibody (Santa Cruz). Samples were washed 3 times for 5 min in PBS, and then incubated with Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen) for 1 h. Nuclei were counterstained with DAPI (0.2 μg/ml) for 10 min. The stained cells were then analyzed under a fluorescence microscope (Carl Zeiss, Göttingen, Germany) with a 63× objective (oil immersion, aperture 1.3). Fifty nuclei from each experiment were counted and evaluated. All samples were examined in three independent experiments.
9
Immunofluorescence Staining of Transfected Cells
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N2a cells were transfected with indicated plasmids with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. At 48 h post transfection, the cells were washed three times with cold PBS and fixed with 4% (v/v) paraformaldehyde for 10 min at room temperature then washed three times with cold PBS. And the cells were permeabilized in PBS containing 0.5% Triton X-100 for 5 min at 4 °C, then blocked in 10% goat serum which were diluted with PBS for 2 h at 37 °C, and probed with primary antibodies which were diluted with PBS and 5% (w/v) BSA for 2 h at 37 °C. The primary antibodies were against Flag tag (MBL, M185-3L, 1:500) and LAMP-1 (Abcam, Ab208943, 1:100), then treated with Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen, A11012, 1:500) or Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen, R37120, 1:500) as a secondary antibody for 1 h at 37 °C, and then stained with DAPI for 10 min. Cells were again washed three times with PBS, and then imaged with a ZEISS confocal microscope under oil objective.
10
Immunofluorescence Assay for DNA Damage Markers
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LAPC4-KD cells (1 × 106 cells/well) were plated on glass coverslips in 6-well plates. After treatment, cells were washed and fixed for immunofluorescence staining as described previously [32 (link)]. Briefly, the prepared samples were probed with phospho-histone-γ-H2AX antibody (Ser139) (Millipore) and p53-binding protein 1 (53BP1) antibody (Santa Cruz) for 2h followed by incubated with Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen) for 1 h. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (0.2 μg/ml) for 10 min. The stained cells were analyzed under a fluorescence microscope (Carl Zeiss, Göttingen, Germany) with a 63× objective (oil immersion, aperture 1.3).
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