Long description A non-isotopic immunofluorescence staining for the localization of DNA synthesis and cell proliferation. Evaluation of cell cycle progression is essential for investigations in many scientific fields. Measurement of [3H] thymidine incorporation as cells enter S phase has long been the traditional method for the detection of cell proliferation. Subsequent quantification of [3H] thymidine is performed by scintillation counting or autoradiography. This technology is slow, labor intensive and has several limitations including the handling and disposal of radioisotopes and the necessity of expensive equipment. A well-established alternative to [3H] thymidine uptake has been demonstrated by numerous investigators. In these methods bromodeoxyuridine (BrdU), a thymidine analog, replaces [3H] thymidine. BrdU is incorporated into newly synthesized DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, BrdU is detected immunochemically allowing the assessment of the population of cells which are actively synthesizing DNA. Exalpha Biologicals BrdU Immunofluorescence Kit involves incorporation of BrdU into proliferating cells, in vivo or in vitro, and fl uorescent staining of these cells which is achieved using a biotinylated anti-BrdU antibody followed by Streptavidin-FL Conjugate
Antibody come from n/a
Other description The following is the list of components which are included in the BrdU Immunofl uorescence Kit. 1A** 4x Trypsin Enzyme Concentrate 1B** Trypsin Dilution Buffer 12 ml 2 Denaturing Solution 6 ml 3 Blocking Buffer 6 ml 4 Detector Antibody (Biotinylated and pre-diluted) 6 ml 5 Streptavidin-FL Conjugate (pre-diluted) 6 ml 6 Mounting Media 6 ml 7 5 Control Slides: Intestinal tissue 1 Positively from mouse injected with BrdU Stained 4 unstained *The material in this kit is suffi cient to run 50 slides. The average test area is defi ned as a circle around the tissue with an approximate diameter of 2 centimeters. ** Trypsin is only required if using formalin fi xed tissues. If the tissues are fi xed in alcohol, trypsin digestion is not required.
Antibody's reacts with these species This antibody doesn't cross react with other species
Antibody's specificity Preparation of Slides Paraffin-Embedded tissue sections: 1. Sample animals are labeled with BrdU 2. Animals are sacrifi ced by inhalation of isofluorane and perfused with PBS followed by 4% buffered formalin. 3. Target tissue is removed and immersed in 4% buffered formalin over night. 4. Tissue is then dehydrated and embedded in paraffin a. PBS – 10 min b. 70% EtOH – 1 hour c. 85% EtOH – 1 hour d. 95% EtOH – 30 min e. 100% EtOH – 15min (2X) f. Xylene – 15 min (2X) g. 1:1 Xylene and Paraffin – 45 min h. Paraffi n – 30 min (4X) 5. 5 micron sections are cut from the paraffin blocks and placed on slides. 6. Slides remain on a 37ºC heating tray overnight and are then stored at 4ºC. Cultured Cells and Cell Suspensions Preparation of Cells A. Cells in Flasks 1. Using sterile tissue culture techniques, culture cells with 10μM BrdU for 2-24 hours at 37ºC. 2. Remove the media containing the BrdU label and wash twice with PBS. 3. Using a cytospin, centrifuge 100μl of cells at 1x 106 cells/ml onto suitable slides and allow to air dry. B. Cells on Chamber Slides (Adherent cells only) 1. Using sterile tissue culture techniques, culture cells in chambers with 10μM BrdU for 2-24 hours at 37ºC. 2. Remove the labeling media and wash twice with PBS. 3. Fix cells with 70% ethanol or other suitable fixative for 30 minutes. 4. Wash twice with PBS. Proceed with Staining Protocol
Antibody's suited for Staining Protocol 1. Deparaffinization (FOR PARAFFIN-EMBEDDED TISSUES ONLY) Note: If you are not using paraffi n-embedded tissues, skip to step 2 below. If paraffi nembedded tissues are used, it is necessary to deparaffinize the slides before following the BrdU staining protocol below. Deparaffi nization involves incubation of the slides in xylene followed by a graded alcohol series as follows: Xylene 5 Minutes, then change to new coplin jar containing Xylene Xylene 5 Minutes 100% ethyl alcohol 5 Minutes 90% ethyl alcohol 3 Minutes 80% ethyl alcohol 3 Minutes 70% ethyl alcohol 3 Minutes PBS 3 Minutes 2. Staining Component: Component 10 (not provided) Component Preparation: Quenching Solution (not provided). Dilute 30% hydrogen peroxide* 1:10 in methanol. Procedure: Immerse slides into a coplin jar or other appropriate container filled with quenching solution for 10 minutes. Wash with PBS 1x for 2 minutes. Time (min): 10 Component: Component 1A and 1B Component Preparation: Trypsin (0.2% solution)** FOR FORMALIN FIXED TISSUES ONLY. Add 1 drop of Component 1A to 3 drops of Component 1B and mix well. Procedure: Add 2 or more drops to each slide. Incubate at room temperature for 10 minutes, followed by a 3 minute rinse in distilled water. Time (min): 10 Component: Component 2 Component Preparation: Denaturing Solution Procedure: Add 2 or more drops to each slide and incubate at room temperature for 30 minutes. Wash twice with PBS, 2 minutes per wash. Time (min): 30 Component: Component 3 Component Preparation: Blocking solution Procedure: Add 2 or more drops to each slide and incubate at room temperature for 10 mins. Drain the solution by blotting on paper towels (DO NOT RINSE). Time (min): 10 Component: Component 4 Component Preparation: Detector Antibody Procedure: Add 2 or more drops to each section and incubate at room temperature for 60 minutes. Wash twice with PBS, 2 minutes per wash. Time (min): 60 Component: Component 5 Component Preparation: Streptavidin-FL conjugate Procedure: Add 2 drops or more to each section and incubate at room temperature for 10 minutes. Wash twice with PBS, 2 minutes per wash. Time (min): 10 Component: Component 6 Component Preparation: Mounting Media Procedure: Add 1-2 drops of mounting media and coverslip. Time (min-) Analyze slide(s) using a fl uorescence microscope, excitation 556, emission * Hydrogen peroxide is not stable for long periods of time. Be sure the reagent you are using has not expired. ** The concentration of trypsin used is very important. It may be necessary to titer the trypsin reagent for use in your system. Usually a final concentration of 0.02% to 0.2% is appropriate. Other methods for digesting the tissue to expose epitopes for antibody recognition may also be used.
Storage The Exalpha Biologicals BrdU Immunofl uorescence Kit components are shipped on blue ice. Upon receipt, store entire kit at -20oC. Once the kit is thawed, you may keep at 4ºC for 5 days. For long-term storage, it is recommended you aliquot and freeze the components at -20ºC, particularly the Streptavidin-FL Conjugate, the Detector Antibody and the Trypsin Concentrate. Wearing of latex or rubber gloves is recommended when running this kit.
Relevant references no information yet
Protein number see ncbi
Warnings The Exalpha Biologicals BrdU Immunofluroescence Kit is a histochemical staining kit for the detection and localization of bromodeoxyuridine incorporated into newly synthesized DNA of actively proliferating cells. This assay is for research use only and not for use in diagnostic or therapeutic procedures.
Test Immunofluorescence kits are used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies.