The cells were fixed and permeabilized using PerFix EXPOSE kit (Beckman Coulter) followed by staining with specific antibodies: mouse monoclonal IgG2b anti-BRCA1 (#MAB22101, R&D Systems), mouse monoclonal IgG1 anti-BRCA2 (#MAB2476, R&D Systems) and rabbit polyclonal anti-PALB2 (#A301-246, Bethyl Laboratories) conjugated with fluorochromes (Alexa Fluor-405, Alexa Fluor-488 or allophycocyanin) using Zenon labeling kit (Life Technologies). Isotype control antibodies (Abcam) were used as controls. Flow cytometry analysis was performed using LSR Fortessa (Beckton Dickinson).
Zenon labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Zenon labeling kit is a set of reagents designed for the fluorescent labeling of antibodies. The kit provides a simple and efficient method for conjugating antibodies with a variety of fluorescent dyes, enabling their use in various immunoassay and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Isotype control antibody, by Abcam (1 mentions)
Perfix expose kit, by Beckman Coulter (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Liberase, by Roche (1 mentions)
Cd103 apc, by Thermo Fisher Scientific (1 mentions)
Cd64 percpcy5, by BioLegend (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Ecl plu, by Cytiva (1 mentions)
A5316, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Promega (1 mentions)
Bz 8000, by Keyence (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
13 protocols using zenon labeling kit
1
Characterizing BRCA and PALB2 Protein Levels
2
Immunostaining of the Placozoan Trichoplax
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T. adhaerens were left to adhere at the bottom of 35-mm plastic Petri dish filled with ASW, swiftly replaced by 4% paraformaldehyde (PFA) with Dextran in ASW for fixation (1-2 hr, RT). After brief washes in ASW followed by 0.1 M phosphate buffer pH7.4 (PB) and a 1 hr-blocking step in 3% normal goat serum (NGS) + 0.2% Triton X-100 in PB, they were incubated for 2 hr in primary antibody (antipeptide: 1 mg/mL; anti-FMRFamide (1:300; Enzo Life Sciences #BML-FA1155 or Phoenix Pharmaceuticals #H-047-29) in blocking solution. After thorough washes in PB, a 1-hr incubation in the corresponding secondary antibody coupled to a fluorophore (1:600, GAR-Alexa Fluor, Molecular Probes) and further washes in PB, they were incubated for 2 min in Hoechst (1:10,000; Molecular Probes), rinsed and mounted with Prolong-Gold (LifeTech) on glass slides. For co-labeling with antibodies raised in the same species, T. adhaerens were instead incubated 45 min in a mix of antibodies coupled to Alexa fluorophores using the Zenon labeling kit (LifeTechnologies) following the manufacturer's instructions, washed in PB, post-fixed 15 min in 4% PFA, washed again and incubated in Hoechst before being mounted on a slide. For blocking experiments, we pre-incubated the antibodies in three times excess of the respective peptides for 2 h before immunostainings.
3
Labeling Neuronal GABAA Receptor Subunits
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies against extracellular epitopes of the GABAAR γ2 or β2,3 subunits (rabbit: 1:500; Synaptic Systems, Gottingen, Germany; mouse: 1:100; Millipore) were prepared in phosphate buffered saline (PBS) at the predetermined concentration. The primary antibodies were labeled with fluorescent Fab’ fragments (Zenon labeling kit; Invitrogen) according to the manufactures instructions (Zenon 647nm—rabbit and Zenon 488—mouse, respectively). At 14–18 DIV, primary hippocampal cultures were incubated for 1 hour at 37°C with the primary/Fab’ mixtures (antibodies against GABAAR γ2 or β2,3 subunits) or a primary antibody against Vesicular GABA transporter (VGAT) lumenal domain fluorescence-labeled with Oyster 488 (rabbit: 1:200; Synaptic Systems [42 (link)]). After the incubation period, the cells were gently washed three times in physiological solution ("Tyrode's", 119mM NaCl, 2.5mM KCl, 2mM CaCl2, 25mM HEPES, 30mM glucose, buffered to pH 7.4) and imaged immediately.
4
Isolation and Identification of Lung and MLN Cells
5
Immunofluorescence Analysis of HEK293T and ALI Cells
6
Quantifying Autophagy Markers via Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the expression of LC3, the cells were fixed with 4% paraformaldehyde. The fixed cells were stained with anti-LC3 antibodies (M152-3; MBL Co. Ltd., Nagoya, Japan). Labeling was performed with the Zenon® labeling kit (Molecular Probes, Inc., Eugene, OR, USA). A fluorescence microscope (BZ-8000; Keyence, Osaka, Japan) was used for observation.
Total protein was extracted and quantified with a protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies for LC3 and β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA) followed with horseradish peroxidase-conjugated anti-mouse IgG (Promega, Madison, WI, USA), and enhanced chemiluminescence (ECL plus; Amersham Life Science, Amersham, UK), as per the manufacturer’s instructions.
Total protein was extracted and quantified with a protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies for LC3 and β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA) followed with horseradish peroxidase-conjugated anti-mouse IgG (Promega, Madison, WI, USA), and enhanced chemiluminescence (ECL plus; Amersham Life Science, Amersham, UK), as per the manufacturer’s instructions.
7
Immunofluorescence Microscopy of HHV-8 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence microscopy was done on HHV-8-infected or uninfected cells by using an anti-mouse MAb directed against ORF-K8.1A/B (ABI) as previously described (12 (link)). Briefly, cells were counted, spotted onto poly-l -lysine-coated slides, fixed in 4% paraformaldehyde for 20 min, and permeabilized with buffer for 20 min at room temperature. Cells were incubated with the K8.1A/B MAb directly conjugated with phycoerythrin or Texas Red by using a Zenon labeling kit (Molecular Probes). Cells were also stained by direct immunofluorescence with an FITC-conjugated anti-CD209 MAb (clone 120507; R&D Systems) or anti-CD207 (clone DCGM4 or clone 343828). To avoid nonspecific binding of IgG, a blocking step was added by using SuperBlock blocking buffer (Pierce). Uninfected cells and cells stained with isotype controls were used as controls. Cells were visualized at a ×60 magnification with a Nikon Eclipse E600 microscope.
8
Immunohistochemical Analysis of Vaccine Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Skin biopsies were performed 24 hours post-injection and embedded in optimal cutting temperature compound (OCT) and frozen in liquid nitrogen. Ten micrometer sections were fixed in 4% of Paraformaldehyde (PFA) for 15 min at room temperature. Tissue was permeabilized with Triton X-100 (0.3%), Bovine serum albumin (BSA) (1%) and goat serum (1%). Skin sections were incubated overnight at 4 °C with 10 µg/ml of anti-GFP-AF488 Ab (Invitrogen, France) or anti-p24 Ab (kind gift from B. Verrier, UMR 5086 CNRS/UCBL, Lyon, France) to detect the expression of vaccine antigens in animals injected with the auxo-GTU®-Luc-EGFP or the auxo-GTU®-multiHIV plasmid, respectively. For keratinocyte staining, an additional incubation for 2 hours was performed with an anti-cytokeratin Ab (clone AE1/AE3, Dako, France). Purified antibodies were labeled with the Zenon® labeling kit (Molecular Probes, Invitrogen). The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) or Propidium Iodide (Invitrogen, France).
9
Immunocytochemical Analysis of Glial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mixed glial cells prepared from neonatal Wistar rat brains (P1) were plated on eight‐well Lab‐Tek chamber slides (Nalge Nunc International) and cultured for 10 days. Rat type 1 MG, type 2 MG, and peritoneal Mφ were plated on the chamber slides and cultured for 1 day. The cells were then incubated with 9F5 (1 µg ml−1; mouse IgG1), rabbit anti‐Iba1 (2.5 µg ml−1; Wako), or the isotype matched controls. Alexa Fluor 488‐ or 594‐labeled second antibody (1:500; Molecular Probes) was used. For double‐staining with 9F5 and anti‐lysosomal‐associated membrane protein‐1 (LAMP‐1; 1 µg ml−1; Ly1C6, mouse IgG1;Stressgen Bioreagents), the Zenon labeling kit (Molecular Probes) was used. Stained cells were observed with a confocal laser scanning microscope (Fluoview, Olympus).
10
Immunofluorescent Staining of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incubation with 0.1% sodium cyanoborohydride and 0.3 M glycine (Sigma-Aldrich), fixed brain slides were incubated with primary antibody for 1 hour at RT. Then, slides were washed in buffer with 0.05% Tween and stained with an AlexaFluor antimouse secondary antibody (Molecular Probes, Eugene, OR) for 30 minutes at RT. Incubation was performed in buffer with 10% normal human serum. For double staining, the second primary antibody was either detected with Alexa-Fluor antibody (for rabbit anti-glial fibrillary acidic protein [GFAP]) or directly labeled to an AlexaFluor using a Zenon labeling kit (Molecular Probes, for mouse anti-CD68 and anti-NeuN mAbs). Slides were washed with buffer and incubated with 0.3% Sudan Black B (Sigma-Aldrich) to prevent background fluorescence by lipids. Nucleus staining was performed with DAPI (4',6-diamidino-2-phenylindole; Life Technologies, Schiphol-Rijk), and the slides were embedded with Vectashield (Vector laboratories, Burlingame, CA). We used a fluorescence (Zeiss Axioplan 2) and confocal laser-scanning (Leica TCS SP2) microscope to analyze the cells.
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!