Antibody to BrdU - thymidine analogue incorporated into DNA during replication therefore used as a marker of proliferating cells. Part of the ValidAb™ range of highly validated, data-rich antibodies.
Figure 1. BrdU Immunoreactivity in HEK293T cells either serum starved or starved then stimulated with 20% serum.
Serum starvation inhibits cell proliferation resulting in no BrdU staining due to a lack of DNA replication. Serum stimulation of starved cells causes cell cycle resumption and DNA replication therefore strong BrdU staining. Method: HEK293T cells were cultured on coverslips in 10% FBS, 1% pen/strep in DMEM until approximately 50% confluency. Cells were then cultured in serum free media for 24 hours. 10µm BrdU (HB0979) was added to all coverslips then half were incubated in 20% FBS containing media for 2hrs while the remainder were kept in DMEM. Cells were then fixed in 4% PFA before being permeabilised with 0.1% Triton X-100 and blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C at a concentration of 1µg/ml (1:1000 dilution). Following washing, a goat polyclonal anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution) was incubated for one hour. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. Images were all taken using the same settings for each condition (40x objective, 0.44µm z-spacing, 405nm: 24.5% power @ 575V gain, 488nm: 23.4% power @ 768V gain). Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 2. BrdU staining reveals proliferation in HEK293T cells using HB9919
Strong BrdU immunoreactivity can be seen caused by the rapid proliferation of HEK293T cells in culture leading to high levels of BrdU integration during DNA replication. Method: HEK293T cells were cultured on coverslips in 10% FBS, 1% pen/strep in DMEM in the presence of 10µm BrdU (HB0979). When cells reached confluency they were fixed with 4% PFA before being permeabilised with 0.1% Triton X-100 and blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C at a concentration of 2µg/ml (1:500 dilution). Following washing, a goat polyclonal anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution) was incubated for one hour. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 40x objective, 0.44µm z-spacing, 405nm (24.5% power @ 575V gain) and 488nm (23.4% power @ 768V gain) lasers. Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 3. Mixed proliferating and non-proliferating cells in mixed neuronal cultures visualised using HB9919.
BrdU staining can be seen amongst the proliferating cells found within mixed neuronal cultures while cells not actively dividing are unstained. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (2µg/ml, 1:500 dilution) followed by a one hour incubation with a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DMi8 inverted epifluorescence microscope connected to a Photometric Prime95B camera. The image was taken using a 40x objective and 1.0µm z-spacing with DAPI (95ms exposure) and L5 (112.7ms exposure) filters. Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to subtract background and perform a maximum Z projection.
Figure 4. Proliferating cell in mixed neuronal cultures visualised with HB9919 staining.
A proliferating cell within mixed neuronal cultures is visualised having incorporated BrdU into its DNA. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (0.5µg/ml, 1:2000 dilution) followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 63x objective, 0.28µm z-spacing, 405nm (29.0% power @ 598V gain), 488nm (1% power @ 907V gain) and 532nm (38.0% power @ 702V gain) lasers. Following capture, deconvolution was carried out using Huygens Professional (SVI) then ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 5. Proliferating cell in mixed neuronal cultures visualised with HB9919 staining.
A proliferating cell within mixed neuronal cultures is visualised having incorporated BrdU into its DNA. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (0.25µg/ml, 1:4000 dilution) followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 63x objective, 0.24µm z-spacing, 405nm (29.0% power @ 663V gain), 488nm (1% power @ 938V gain) and 532nm (30.1% power @ 685V gain) lasers. Following capture, deconvolution was carried out using Huygens Professional (SVI) then ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 6. Concentration response of HB9919 staining in mixed neuronal cultures.
HB9919 identifies proliferating cells in mixed neuronal cultures at dilutions as low as 1:4000 (0.25µg/ml) while not showing any cross-reactivity in the absence of BrdU. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated at 4°C overnight in dilutions ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml). This was followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMi8 inverted epifluorescence microscope (63x objective) connected to a Photometric Prime95B camera. Exposure settings were as follows:
1:500 (no BrdU) – DAPI_LP: 40.1ms, FITC_LP: 361ms, RHOD_LP: 149.3ms
Following capture ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to subtract background and perform a maximum Z projection.
Figure 1. BrdU Immunoreactivity in HEK293T cells either serum starved or starved then stimulated with 20% serum.
Serum starvation inhibits cell proliferation resulting in no BrdU staining due to a lack of DNA replication. Serum stimulation of starved cells causes cell cycle resumption and DNA replication therefore strong BrdU staining. Method: HEK293T cells were cultured on coverslips in 10% FBS, 1% pen/strep in DMEM until approximately 50% confluency. Cells were then cultured in serum free media for 24 hours. 10µm BrdU (HB0979) was added to all coverslips then half were incubated in 20% FBS containing media for 2hrs while the remainder were kept in DMEM. Cells were then fixed in 4% PFA before being permeabilised with 0.1% Triton X-100 and blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C at a concentration of 1µg/ml (1:1000 dilution). Following washing, a goat polyclonal anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution) was incubated for one hour. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. Images were all taken using the same settings for each condition (40x objective, 0.44µm z-spacing, 405nm: 24.5% power @ 575V gain, 488nm: 23.4% power @ 768V gain). Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 2. BrdU staining reveals proliferation in HEK293T cells using HB9919
Strong BrdU immunoreactivity can be seen caused by the rapid proliferation of HEK293T cells in culture leading to high levels of BrdU integration during DNA replication. Method: HEK293T cells were cultured on coverslips in 10% FBS, 1% pen/strep in DMEM in the presence of 10µm BrdU (HB0979). When cells reached confluency they were fixed with 4% PFA before being permeabilised with 0.1% Triton X-100 and blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C at a concentration of 2µg/ml (1:500 dilution). Following washing, a goat polyclonal anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution) was incubated for one hour. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 40x objective, 0.44µm z-spacing, 405nm (24.5% power @ 575V gain) and 488nm (23.4% power @ 768V gain) lasers. Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 3. Mixed proliferating and non-proliferating cells in mixed neuronal cultures visualised using HB9919.
BrdU staining can be seen amongst the proliferating cells found within mixed neuronal cultures while cells not actively dividing are unstained. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (2µg/ml, 1:500 dilution) followed by a one hour incubation with a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica DMi8 inverted epifluorescence microscope connected to a Photometric Prime95B camera. The image was taken using a 40x objective and 1.0µm z-spacing with DAPI (95ms exposure) and L5 (112.7ms exposure) filters. Following capture, ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to subtract background and perform a maximum Z projection.
Figure 4. Proliferating cell in mixed neuronal cultures visualised with HB9919 staining.
A proliferating cell within mixed neuronal cultures is visualised having incorporated BrdU into its DNA. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (0.5µg/ml, 1:2000 dilution) followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 63x objective, 0.28µm z-spacing, 405nm (29.0% power @ 598V gain), 488nm (1% power @ 907V gain) and 532nm (38.0% power @ 702V gain) lasers. Following capture, deconvolution was carried out using Huygens Professional (SVI) then ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 5. Proliferating cell in mixed neuronal cultures visualised with HB9919 staining.
A proliferating cell within mixed neuronal cultures is visualised having incorporated BrdU into its DNA. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated overnight at 4°C (0.25µg/ml, 1:4000 dilution) followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was taken using a 63x objective, 0.24µm z-spacing, 405nm (29.0% power @ 663V gain), 488nm (1% power @ 938V gain) and 532nm (30.1% power @ 685V gain) lasers. Following capture, deconvolution was carried out using Huygens Professional (SVI) then ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to perform a maximum Z projection.
Figure 6. Concentration response of HB9919 staining in mixed neuronal cultures.
HB9919 identifies proliferating cells in mixed neuronal cultures at dilutions as low as 1:4000 (0.25µg/ml) while not showing any cross-reactivity in the absence of BrdU. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498). 10µM BrdU was added to cultures on DIV7 before cells were fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by incubation with 2M HCl for 20 minutes at 37°C. Cells were incubated in 0.1M borate buffer (pH8.5) for 10 minutes before being blocked in 1% BSA, 300mM glycine. HB9919 was incubated at 4°C overnight in dilutions ranging from 1:500 (2µg/ml) to 1:4000 (0.25µg/ml). This was followed by a one hour incubation with Rhodamine Phalloidin TRITC (HB8621, 183nM) and a polyclonal goat anti-mouse Dylight 488 conjugated secondary antibody (Thermofisher 35503, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMi8 inverted epifluorescence microscope (63x objective) connected to a Photometric Prime95B camera. Exposure settings were as follows:
1:500 (no BrdU) – DAPI_LP: 40.1ms, FITC_LP: 361ms, RHOD_LP: 149.3ms
Following capture ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676-682) was used to subtract background and perform a maximum Z projection.
Product information
Immunogen
BrdU conjugated with hemocyanine.
Clone number
MoBu-1
Isotype
IgG1
Purification
Protein A affinity chromatography
Formulation
Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant albumin
Predicted species reactivity
NA
Tested species reactivity
NA
Tested applications
Applications
ICC, IHC(IF)
IHC(IF) optimal concentration
1µg/ml (1:1000) as measured in rat hippocampus.
ICC optimal concentration
1µg/ml (1:1000) as measured in mixed neuronal cell cultures.
Product specific protocols
The dense structure of chromatin can prevent anti-BrdU antibodies binding to the intercalated BrdU within the DNA helix. Denaturing the DNA can therefore improve staining:
Incubate brain sections or coverslips in 2M HCl for 30 minutes at 37°C
Incubate with 0.1M sodium tetraborate (2 x 5 minute incubations) to neutralise the acid
Wash in PBS / TBS (3 x 5 minute washes)
Continue with immunostaining (see our IHC(IF) and ICC protocols for more information)
50:50 ratio of dH2O to glycerol and storing at -20°C
dH2O then aliquot and store at -80°C
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube.
We sell a range of different pack sizes of BrdU which we have succesfully used with this antibody. Please follow this link to find out more information.
What counterstains do you recommend for use in ICC and IHC with this BrdU antibody?
What protocols are available for use with this BrdU antibody?
We have made a comprehensive collection of protocols that we have used in our experiments to validate this BrdU antibody. Adittionally for using BrdU to measure neurogenesis please refer to Wojtowicz and Kee et al., 2006. for a detailed protocol and troubleshooting.
What guarantee do you have that my BrdU antibody will perform as expected?
We guarantee that your BrdU antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.
Antibody to BrdU - thymidine analogue incorporated into DNA during replication therefore used as a marker of proliferating cells. Part of the ValidAb™ range of highly validated, data-rich antibodies.
Antibody to tyrosine hydroxylase (TH) - the rate limiting enzyme in catecholamine synthesis and used as a marker for catecholaminergic (dopaminergic and noradrenergic) neurones in the CNS. Part of the ValidAb™ range of highly validated, data-rich antibodies.
Western Blot Protocol (1 MB) 
