Pregnant females were intraperitoneally injected with 50 mg/kg BrdU 3 h before being euthanized and having embryos dissected out. Embryos were fixed, sucrose-protected, and frozen-sectioned, antigen-retrieved by heating for 5 min to 85°C in 75% formamide/PBS, and detected with anti-BrdU antibody (1:200 dilution; Abcam, BU1/75 ICR1).
Bu1 75 icr1
Manufactured by Abcam
The BU1/75 ICR1 is a primary antibody that targets the CD3 epsilon chain, a component of the T cell receptor complex. It is commonly used in immunological research to label and identify T cells.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i fluorescence microscope, by Nikon (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Goat anti rat alexafluor 594, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse cy2, by Jackson ImmunoResearch (1 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
5 iodo 2 deoxyuridine idu, by Merck Group (1 mentions)
5 chloro 2 deoxyuridine cldu, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Ecl plus reagent, by PerkinElmer (1 mentions)
Bm chemiluminescence elisa substrate, by BD (1 mentions)
Anti cd9 antibody, by BD (1 mentions)
8 protocols using bu1 75 icr1
1
Embryonic Cell Proliferation Analysis
2
DNA Fiber Analysis of CldU and IdU Incorporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pulse-labelled with 50 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU) as indicated, with or without treatment as reported in the experimental schemes. DNA fibers were spread out as previously reported (26 (link)). For immunodetection of labelled tracks the following primary antibodies were used: anti-CldU (1:60; rat-monoclonal anti-BrdU/CldU; BU1/75 ICR1 Abcam) and anti-IdU (1:10; mouse-monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson). The secondary antibodies were goat anti-mouse Alexa Fluor 488 or goat anti-rat Alexa Fluor594 (Invitrogen). The incubation with antibodies was accomplished in a humidified chamber for 1 h at 37°C. Images were acquired randomly from fields with untangled fibers using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks was measured using the Image-Pro-Plus 6.0 software. A minimum of 100 individual fibers were analyzed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using GraphPad Prism Software.
3
Dual-Label Immunostaining for BrdU and IdU
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to immunostaining, slides were washed twice with ddH2O for 5 min, 1 × 2.5 M HCl, denatured with 2.5 M HCl for 1 h, rinsed twice with PBS and then washed with blocking solution (PBS with 1% BSA and 0.1% Tween-20) twice for 5 min and then for 1 h. For immuno-labelling all antibodies were dissolved in blocking solution. Slides were then incubated with a rat anti-BrdU antibody (BU1/75 [ICR1], 1:500, Abcam cat# 6326; RRID: AB_305426) to detect BrdU for 1 h under humidified conditions, rinsed with PBS (× 3), fixed for 10 min with 1% formaldehyde, rinsed with PBS (× 3), and quenched with glycine. Slides were then rinsed with PBS (× 3) followed by overnight, 4 °C incubation with mouse anti-BrdU (B44, 1:100, BD Biosciences cat#347580; RRID: AB_400326) to detect IdU. Slides were then washed twice with PBS, 3 times for 5 min in blocking solution, followed by incubation in the appropriate fluorescently-conjugated secondary antibodies diluted in blocking solution (1:500; donkey anti-rat Cy3 cat#712–165-153 RRID: AB_2340667; donkey anti-mouse Cy2 cat#715–225-150 RRID: AB_2340826; all Jackson ImmunoResearch Laboratories) for 1.5 h. Post-incubation, slides were washed 2 x PBS, 3 × 5 min with blocking solution and 2 x PBS. All slides were mounted to coverslips using PBS/Glycerol (1:1).
4
DNA Fiber Assay for DNA Replication
5
DNA Fiber Assay for Replication Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pulse-labelled with 25 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU) at specified times, with or without treatment as reported in the experimental schemes. Cells were or not pre-treated with the transcription inhibitor 5,6-dichloro-1-ß-d -ribofurosylbenzimidazole (DRB 50 μM for 1 h). DNA fibres were prepared and spread out as previously reported (35 (link)). For immunodetection of labelled tracks the following primary antibodies were used: anti-CldU (rat-monoclonal anti-BrdU/CldU; BU1/75 ICR1 Abcam) and anti-IdU (mouse-monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson). The secondary antibodies were: goat anti-mouse Alexa Fluor 488 or goat anti-rat Alexa Fluor 594 (Molecular Probes). The incubation with antibodies was accomplished in a humidified chamber for 1 h at RT.
Images were acquired randomly from fields with untangled fibres using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks were measured using the Image-Pro-Plus 6.0 software, and values were converted into kilobases using the conversion factor 1 μm = 2.59 kb as reported (35 (link)). A minimum of 100 individual fibres were analysed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using Graph Pad Prism Software.
Images were acquired randomly from fields with untangled fibres using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks were measured using the Image-Pro-Plus 6.0 software, and values were converted into kilobases using the conversion factor 1 μm = 2.59 kb as reported (35 (link)). A minimum of 100 individual fibres were analysed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using Graph Pad Prism Software.
6
Analyzing DNA Replication Using Southern Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total intracellular DNA was extracted from SV40- and mock-infected cells and subjected to agarose gel electrophoresis and Southern blotting as described [30] (link). In Figure 1 , the same amount of SV40 DNA was loaded into each lane. In all other Southern blots, loading was normalized to cell number. In both cases, mitochondrial DNA was probed as an internal loading control. Loading determined by cell number or by mitochondrial DNA signal was highly correlated. 2D gel electrophoresis was as described by [30] (link) with the following modification: the second dimension of the 2D gel was run for 7 h through a 0.95% 1×TBE agarose gel containing 0.5 ng/mL ethidium bromide. Southern blotting probes and data analysis using ImageQuant 5.2 were as previously described [30] (link).
For DNA extractions from cells exposed to 20 µM EdU for 30 minutes (Figure 6A ), DNA was isolated as per [30] (link), but dissolved in 1 µL of 10 mM Tris pH 8.0 with 0.1 mM EDTA per 20,000 cells. A rat anti-BrdU antibody ([BU1/75 (ICR1)], Abcam), anti-rat conjugated to HRP (Jackson ImmunoResearch), and ECL-plus reagent (Perkin Elmer) were used to detect EdU label on the Southern blot as described in [83] (link).
For DNA extractions from cells exposed to 20 µM EdU for 30 minutes (
7
Indirect and Direct ELISA for sEV Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the indirect sandwich ELISA, an anti-BrdU antibody (BU1/75 (ICR1), 5 µg/mL, Abcam, catalogue no. ab6326) was coated on a black 96-well plate and incubated overnight at 4°C. Plates were blocked with 1% BSA in PBS for 1 h at RT. For each sEV sample, 100 µg were added to the plate and incubated for 2 h at RT. Next, anti-CD9 antibody (p24, 1 µg/mL, BD Biosciences, catalogue no. 555370) was added to the samples and incubated for 1 h at RT. After washing three times for 5 min with PBS, HRP-conjugated anti-mouse antibody was incubated with the samples for 1 h. Luminescent signal was measured with the BM Chemiluminescence ELISA Substrate (BD Biosciences) as recommended by the manufacturer. For the direct ELISA, 100 µg of sEVs were coated on 96-well plates overnight at 4°C. Plates were blocked with 1% BSA in PBS for 1 h and incubated with anti-CD9 antibody (BD Biosciences) for 2 h at RT. After washing three times for 5 min with PBS, an HRP-conjugated secondary antibody was incubated with the samples for 1 h at RT, and the luminescent signal was measured using a Varioskan instrument.
8
BrdU Incorporation Assay for Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before the mechanical stress stimulation, the culture medium was refreshed with new medium containing 10 μM bromodeoxyuridine (BrdU). Following 8 h of stimulation, the cells were fixed with 4% PFA, then incubated with 2 N HCl solution for 30 min, and subsequently incubated with 0.1 N sodium borate for 20 min at room temperature. Then, the cells containing BrdU were immunostained following the procedure mentioned above using the primary antibody anti-BrdU [BU1/75 (ICR1)] (1:250, Abcam) and the secondary antibody rabbit anti-rat IgG (Alexa Fluor 488). The cell proliferation state was expressed as the ratio BrdU-positive cell number/DAPI-positive cell number.
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!