For HA-SENP1, HA-SENP2 staining, cultured U2OS cells were transient transfected with above HA tagged plasmids. Cells were stained with anti-HA antibody (cat. #H6908, Sigma). For endogenous SENP2 staining, cells were stained with anti-SENP2 antibody (cat. # ab58418, Abcam). For endogenous PIAS4 and CENP-A staining, U2OS cells were stained with anti-PIAS4 (cat. # ab211625, Abcam) and anti-CENP-A (cat. #2186, Cell Signaling). After incubation with the primary antibodies at room temperature for 1 hr, Alexa 488- and Alexa 594-labeled secondary antibodies (Life Technology) were added for 1 hr. Cells were then adhered to a slide with DAPI-staining Mounting Medium (Vector Labs). All samples were visualized with an Olympus fluorescence microscope and images were derived with the accompanying DP-BSW application software program.
Alexa 594 labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-labeled secondary antibodies are fluorescent detection reagents used in immunoassays and other biological applications. These antibodies specifically bind to primary antibodies, allowing for the visualization and localization of target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (3 mentions)
Streptavidin alexa 488, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Anti cenpa, by Abcam (1 mentions)
Dapi staining mounting medium, by Vector Laboratories (1 mentions)
Cd11b, by BD (1 mentions)
Neun antibody, by Merck Group (1 mentions)
Col2a1, by Abcam (1 mentions)
Primary antibodies against γ h2ax, by Cell Signaling Technology (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lsr 2 flow cytometer system, by BD (1 mentions)
Eclipse te200, by Nikon (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Fv1000 confocal system, by Olympus (1 mentions)
Hoechst, by Merck Group (1 mentions)
Tcs sp8, by Leica (1 mentions)
P pak4 ser474, by Cell Signaling Technology (1 mentions)
17 protocols using alexa 594 labeled secondary antibody
1
Immunofluorescence Staining of HA-SENP1, HA-SENP2, Endogenous SENP2, PIAS4, and CENP-A in U2OS Cells
2
Retinal Immunohistochemistry of p-STAT3
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Immunohistochemical analysis of p-STAT3 was performed as previously reported [21] (link). Mice were perfused 1 hour after intravitreal injection with OSM (3 µg, 2 µL). The retinas were fixed in 4% paraformaldehyde and cryoprotected by 30% sucrose in 0.1 M phosphate buffer. Cryo-sections (16 µm) were stained with antiphospho-STAT3 (phosphor Tyr705) antibodies (Abcam, Cambridge, MA) and visualized using the TSA signal amplification kit (Life Technologies, Grand Island, NY) according to manufacturers’ instructions. Double staining was carried out with either NeuN antibodies (EMD Millipore, Billerica, MA) for RGC, or anti-glutamine synthetase antibodies (EMD Millipore) for Müller cell identification.
To identify microglial cells in the retina after ON-crush, retrograde labeling with FG was performed as described above, followed by 4-s ON-crush 7 days after FG labeling. Retinas were harvested 14 days after ON-crush and stained with antibodies against CD11b (BD Biosciences, San Diego, CA) and Alexa 594 labeled secondary antibodies (Life Technologies, Grand Island, NY).
To identify microglial cells in the retina after ON-crush, retrograde labeling with FG was performed as described above, followed by 4-s ON-crush 7 days after FG labeling. Retinas were harvested 14 days after ON-crush and stained with antibodies against CD11b (BD Biosciences, San Diego, CA) and Alexa 594 labeled secondary antibodies (Life Technologies, Grand Island, NY).
3
Evaluating Tight Junction Permeability in Mice
4
Evaluating Tight Junction Permeability in Mice
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To test the TJ barrier permeability in mice, 10 mg/ml EZ-Link sulfo-NHS-LC-biotin (Pierce Chemical) in PBS containing 1 mM CaCl2was intradermally injected underneath the dermis of the E18.5 mice back skin. After 30 minutes of incubation at 37°C, mice back skin was isolated, embedded in Tissue-Tek, and frozen. 4% Paraformaldehyde fixed section was immunostained with TJ marker, anti-Occludin antibody. After washing step, Alexa 594labeled secondary antibodies (Life technologies)for TJ marker and Alexa 488 Streptavidin (Life technologies)for biotin labeling were applied for 30 minutes at RT. Followed by another washing and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen).Image were taken using an Olympus BX43.
5
Detecting DNA Damage in Knee Joints
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The knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against γH2AX (Cell Signaling Technology, Danvers, MA, 1:100) and Col2a1(Abcam, Cambridge, UK). Then, the sections were incubated with species-matched Alexa-488 or Alexa-594-labeled secondary antibodies (Life Technologies, Grand Island, NY, USA). Subsequently, the sections were mounted with PBS containing DAPI (Thermo Fisher Scientific), and photographed using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). The proportions of positively stained cells relative to the total number of cells were determined for each sample.
6
Murine Bone Marrow Cell Phenotyping
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Murine bone marrow cells were extracted and washed with 10 mM TDG or sucrose for 15 min at 4°C. Flow cytometry was performed as previously described (Isaac et al., 2013 (link)). Briefly, after 20 min of fixation with 2% p-formaldehyde (PFA), cells were incubated on ice with 0.5% BSA for 30 min, followed by incubation with galectin-8 antibodies for 1 hr and Alexa 594-labeled secondary antibodies (Life Technologies) for 30 min. Cells were washed with cold phosphate-buffered saline (PBS) between incubations. Flow cytometry analysis was performed by LSR II Flow Cytometer System (BD Biosciences).
7
Immunofluorescent Analysis of hUCB-MSCs
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After the indicated treatments, hUCB-MSCs were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min, permeabilized by incubation with a 0.05% Triton X-100 solution for 10 min and blocked with 5% normal goat serum (NGS) for 1 h. The cells were subsequently stained with specific primary antibodies against COX-2 (Abcam) and NF-κB (Cell Signaling Technology) followed by 2 h of incubation with Alexa 488- and Alexa 594-labeled secondary antibodies (1:1,000; Molecular Probes, Eugene, OR, USA), respectively. For counterstaining, nuclei were stained with DAPI. The images were captured by a confocal microscope (Nikon, Eclipse TE200, Tokyo, Japan).
8
Immunofluorescence Staining Protocol
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Briefly, cells were quickly washed in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed MFE cells or fibroblasts were permeabilized with 0.2% triton-100X for 3 min at room temperature, and blocked with 3% BSA. Primary antibodies were combined with adequate Alexa488 and/or Alexa594-labeled secondary antibodies (Molecular Probes) in PBS with 0.3% BSA. Nuclei were stained with Hoechst (Sigma). Images were acquired using 60X objective in an Olympus FV1000 confocal system. The antibodies used were anti-HA (rat monoclonal 3F10, green), anti-Ccnd1 (rabbit monoclonal EP12 Dako, green) and anti-RalA (mouse monoclonal, red).
9
Immunofluorescence Analysis of Pak4 and p-Pak4
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Cells treated with or without E2 were seeded on coverslips and cultured in DMEM/10% FBS overnight. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with goat serum before incubation with antibodies against Pak4 (1:100, Abcam), or p-Pak4 Ser474 (1:100, Cell Signaling Technology) at 4°C overnight. The cells were incubated with Alexa 594-labeled secondary antibodies (1:200; Invitrogen, Burlington, ON, Canada), and counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime) before analysis using a microscope (Leica TCS SP8). The control slides were treated with PBS instead of the primary antibody.
10
Lung Histology and Immunostaining Protocol
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The left lung was inflated with 4% neutral buffered paraformaldehyde, and the left lung was subsequently removed and placed in fresh 4% neutral buffered paraformaldehyde for 24 h at room temperature. After embedding the tissues in paraffin, they were sliced into 4-µm sections and subjected to H&E and Sirius red staining. Twenty sequential fields of view encompassing the entire lung section were independently and blindly scored by two pathologists with Ashcroft scores as previously described2 (link). For immunostaining, fresh frozen sections (6 μm) were co-incubated with primary antibodies against α-SMA (1:200) or Fsp1(1:100), followed by probing with Alexa 594-labeled secondary antibodies (Invitrogen, Carlsbad, CA, USA). The results were assessed by two pathologists using a fluorescent microscope (Olympus, Japan) in a blinded fashion.
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