The immunostaining assays were performed using a protocol adapted from18 (link). First, brain sections were incubated with 2 M HCl for 25 min at 37 °C to induce DNA denaturation. After washing with PBS, tissue sections were further incubated in a blocking solution containing 2% of horse serum (Life Technologies, Carlsbad, CA, USA) and 0.3% Triton X-100 (Fisher Scientific) diluted in 0.1 M PBS for 2 h at RT. After the blocking procedure, tissue sections were incubated for 72 h at 4 °C in the following primary antibodies (diluted in the blocking solution): rat monoclonal anti-BrdU (1:500, AbD Serotec, Raleigh, NC, USA), goat polyclonal anti-doublecortin (DCX; 1:500, Santa Cruz Biotechnology), or mouse monoclonal anti-NeuN (1:500, Merck Millipore). Then, sections were rinsed in PBS and incubated with Hoechst (1:1000; Sigma-Aldrich Co. LLC) and the respective secondary antibodies: Alexa Fluor-488 donkey anti-rat, Alexa Fluor-546 donkey anti-goat or anti-mouse (all 1:500; all Life Technologies), diluted in a solution containing 0.3% Triton X-100 in 0.1 M PBS, for 2 h at RT. Finally, sections were rinsed in PBS and mounted in Fluoroshield Mounting Medium (Abcam Plc., Cambridge, UK) for further analysis.
Rat monoclonal anti brdu
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Rat monoclonal anti-BrdU is a laboratory reagent used to detect the incorporation of bromodeoxyuridine (BrdU) into cellular DNA. It is a monoclonal antibody derived from rat that specifically binds to BrdU, allowing for the identification and quantification of cells undergoing DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Cy3 conjugated goat anti rat, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Goat polyclonal anti doublecortin dcx, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti neun, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Fluoroshield mounting medium, by Abcam (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Vectashield hard set anti fade reagent, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Nucblue fixed cell stain, by Thermo Fisher Scientific (1 mentions)
Polyclonal rabbit anti gfap, by Agilent Technologies (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Hoechst stain, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
8 protocols using rat monoclonal anti brdu
1
Immunostaining of Neurogenic Markers in Brain Tissue
2
Cell Proliferation and Apoptosis Assay
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To assess cell proliferation, adherent monolayers were cultured on coverslips coated with PDL/laminin in 24-well plates. After treatments, adherent cells were incubated with BrdU (10 μM) for 2 h, then fixed with 4% PFA in 0.1 M PBS for 10 min at RT. For BrdU staining, cells were washed with PBS, and DNA was denatured with 1N HCl for 30 min at 37 °C. For all stainings, cells were blocked for 1 h in blocking solution containing 10% donkey serum and 0.2% Triton X-100 in PBS, followed by an incubation with a primary (rat monoclonal anti-BrdU; 1:500; AbD Serotec); and secondary (Cy3; 1:1000 Jackson ImmunoResearch) antibodies prepared in blocking solution for 2 and 1 h, respectively. The nuclei were labeled with Hoechst and coverslips were mounted onto glass slides for Zeiss Apotome microscope imaging.
To assess apoptosis induction the number of pyknotic nuclei, average nucleus size and integrated density (fluorescence intensity normalized by cell size) were quantified per image using the ImageJ software.
To assess apoptosis induction the number of pyknotic nuclei, average nucleus size and integrated density (fluorescence intensity normalized by cell size) were quantified per image using the ImageJ software.
3
Quantitative Immunofluorescence Analysis of Neurogenesis
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From each brain of group 1, every twelfth section was used for double immunofluorescence with BrdU/DCX, BrdU/GFAP and for Sirt1 immunofluorescence. From each brain of group 2, every twelfth section was stained for BrdU/NeuN and BrdU/GFAP. For BrdU staining, DNA was denatured as described above. Sections were incubated with the following primary antibodies: rat monoclonal anti BrdU (1:500, AbD Serotec, UK) and rabbit polyclonal anti DCX (1:1000, Abcam, England); polyclonal rabbit anti GFAP (1:2000, DAKO, Denmark), polyclonal rabbit anti NeuN (1:1000, Millipore-Chemicon, CA) or polyclonal rabbit anti Sirtuine-1 (Sirt1, 1:1000, Millipore-Chemicon, CA). Sections were then rinsed in PBS, followed by incubation with the following secondary antibodies: Alexa Fluor 488 goat anti rat IgG (1:500, Molecular Probes, USA) and Alexa Fluor 568 goat anti rabbit IgG (1:500, Molecular Probes, USA) for one hour at room temperature. After washing, nuclei were counterstained with NucBlue Fixed Cell Stain (Molecular probes, USA). Sections were then mounted on slides and dehydrated. Finally slides were coverslipped using Vectashield Hard Set anti-fade reagent (Vector Laboratories, CA) and kept in darkness at 4° C.
4
Immunohistochemical Staining Protocols
5
Quantifying Cell Cycle Dynamics in Mice and Geckos
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Both IdU and BrdU were injected (both 70 μg/g body weight) into pregnant ICR mice at E13.5, E15.5 and E16.5 (n = 5) and into juvenile geckos (n = 5). The mandibles of the embryos were embedded in wax and sectioned at 4 μm thickness. We used mouse monoclonal anti‐BrdU (Becton Dickson Ltd., NJ, USA), which can recognize both IdU and BrdU, and rat monoclonal anti‐BrdU (Bio‐Rad, CA, USA). For the secondary antibodies, Alexa Fluor 488‐conjugated goat anti‐mouse (Invitrogen; dilution 1:200) and Alexa Fluor 555‐conjugated goat anti‐rat (dilution 1:200) antibodies were used. The cell cycles were determined according to a previously described formula.20
6
Immunofluorescence Staining for Hippocampal Neurons
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Immunofluorescence staining was performed as described earlier (19) . Free-floating hippocampal brain sections were washed with 0.1 mmol/L phosphate-buffered saline followed by an incubation with 2N hydrochloric acid for 30 minutes at 37°C for antigen retrieval. Tissue sections were then incubated with the primary antibody rat monoclonal anti-BrdU (1:100) (Bio-Rad, Hertfordshire, England) and kept overnight at 4°C. On the 2nd day, tissues were incubated in the dark with the secondary antibody, Alexa Fluor-568 goat anti-rat (Invitrogen; ThermoFisher Scientific, Eugene, Ore), for 2 hours at room temperature. Tissue sections were then incubated with the mouse monoclonal anti-hexaribonucleotide binding protein-3 (NeuN) (1:400) (Millipore, Darmstadt, Germany) overnight at 4°C. On the 3rd day, sections were incubated with the secondary antibody Alexa Fluor-488 goat antimouse (1:250) (Invitrogen; ThermoFisher Scientific) for 2 hours at room temperature. Finally, Hoechst stain (1:10 000) (Invitrogen, ThermoFisher Scientific) was applied to the sections for 10 minutes and hippocampal tissues were mounted on glass slides.
7
BrdU Labeling and Immunohistochemistry
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Mice were administered BrdU (150 mg/kg, i.p. dissolved in saline), 2 hrs before sacrifice in order to assess cell proliferation. For the immunohistochemistry experiments, the sections were washed three times for 10 mins in PBS, mounted onto slides, dried, and then exposed to Citrate Buffer (10 mM Citric Acid, pH 6.0 at 95°C) for 2 hrs. After a brief 1 min wash in PBS, the slides were then incubated overnight at room temperature with the primary antibody (Anti-BrdU rat monoclonal, Serotec, 1:100). The next day, the slides were washed two times for 5 mins with PBS, and were then incubated for 1 hr with the secondary antibody (Cy3-conjugated goat anti-rat, Molecular Probes, 1:200). The slides were then washed three times for 10 min in PBS and coverslipped.
8
BrdU Labeling and Immunohistochemistry
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