BrdU antibody

Catalog No. ABIN2477713

Product Details anti-BrdU Antibody

Target: BrdU (Bromodeoxyuridine (BrdU))

Reactivity: Please inquire

Host: Mouse

Clonality: Monoclonal

Conjugate: This BrdU antibody is un-conjugated

Application: Flow Cytometry (FACS), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunocytochemistry (ICC), Immunohistochemistry (Frozen Sections) (IHC (fro))

Characteristics: Mouse anti BrdU antibody, clone Bu20a, recognizes BrdU and other thymidine analogs; 5′-chloro-2′-deoxyuridine (CldU), 5′-iodo-2′-deoxyuridine (IdU) and 2′-deoxy-5-ethynyluridine (EdU), but only shows minimal reactivity with thymidine itself

Purification: Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant

Immunogen: Bromodeoxyuridine conjugated to BSA

Clone: Bu20a

Isotype: IgG1

Application Details

Application Notes: Flow: 1/25-1/100, ICC 1/25-1/100
For Flow Cytometry use 10 μl of the suggested working dilution to label 1x106 cells in 100 μl

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #103189 (Immunohistochemistry)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #103189

Date: 10/05/2018

Antigen: BrdU

Lot Number: 1710

Method validated: Immunohistochemistry

Positive Control:

Adult (24 months) mouse brain and intestine (duodenum)

Negative Control:

We incubated slices overnight with the blocking solution only and then processed them with the secondary antibody.

Notes:

Passed. ABIN2477713 works well in IHC-P, but with higher concentrations than the ones suggested by the manufacturer.

Validation Images

Staining of mouse brain and intestine sections using ABIN2477713. A-C: A bright staining in the cell nuclei of individual cells scattered across the brain is present, especially in the hippocampus, ventricular wall (A) and sub-cortical areas at the border between the gray and white matters (B-C). Some positive cells are clearly M-phase cells (C). Abbreviations: GM = gray matter; LV = lateral ventricle; WM = white matter. D-E: positive control staining in the intestine (duodenum). BrdU+ proliferating cells are clearly detected in the germinal layer of the intestinal epithelium, at the basis of the duodenal villi.

Full Methods

Primary Antibody: ABIN2477713

Secondary Antibody: goat anti-mouse AF488 conjugated antibody (Thermo Fisher Scientific, A11029, lot 1942237)

Full Protocol:

• Inject mouse intraperitoneally at the dose of 0.1 mg/g body weight with BrdU (Sigma, B5002) dissolved in sterile distilled water at final concentration of 100mg/ml.
• Perfuse mouse was perfused with paraformaldehyde 4% in 0.1M phosphate buffer (PB) pH 7.4 and post-fix samples in the same fixative for an additional 2h at RT.
• Wash, dehydrate, and embed samples in paraffin wax.
• Following several washes in PBS, cut intestines and brain with a microtome (6µm-thick sections) and mount sections on glass slides.
• After paraffin removal, process sections by microwave antigen retrieval for 10min (95-100°C) in 10mM sodium citrate buffer (pH6.0).
• Let sections cool for 20min.
• Wash sections 2x 5min with distilled water.
• Wash sections 5min with PBS.
• Incubate sections in PBS containing 1% albumin from chicken egg white (Sigma, A5378) and 0.3% Triton-X-100 (BioRad, 161-0407, lot 00583) for 1h at RT to block non-specific binding sites.
• Incubate sections with primary antibody mouse anti-BrdU (antibodies-online, ABIN2477713, lot 1710) diluted 1:10 in PBS-BSA-PLL ON at RT.
• Wash sections 3x 5min with 0.01M PBS.
• Incubate sections with the secondary goat anti-mouse AF488 conjugated antibody (Thermo Fisher Scientific, A11029, lot 1942237) diluted 1:200 in 0.1M PBS for 1h at RT.
• Wash sections 3x 5min with 0.01M PBS.
• Mount sections in Fluoroshield (Sigma, F6182, lot MKCB0153V).
• Acquire images with Leica DM 6000B fluorescence microscope equipped with the manufacturer’s FITC filter set and digital camera at magnifications of 20x and 40x. Parameters for image acquisition (Exposure 2.2s, Gain 2.3x, Saturation 1.5, Gamma 1.10) were maintained unchanged for all images.

Experimental Notes:

For the incubation with the primary antibody ABIN2477713 1:25, 1:20, 1:15 dilutions were also tested with also 0.01M PBS - 5% Normal Goat Serum (NGS Sigma, G9023, lot SLBV1396) and 0.1% Triton-X -100 as the blocking solution, but 1:10 and PBS containing 1% albumin from chicken egg white and 0.3% Triton-X gave the best results.

Handling

Format: Liquid

Concentration: 1.0 mg/mL

Buffer: Phosphate buffered saline, 0.09%Sodium Azide (NaN3)

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Handling Advice: This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.

Storage: 4 °C/-20 °C

Storage Comment: Store at +4oC or at -20oC if preferred. Storage in frost-free freezers is not recommended.

Expiry Date: 18 months

References for anti-BrdU antibody (ABIN2477713)

Innis, Dai, Wu, Buchan, Jacobson: "Perinatal lipid nutrition alters early intestinal development and programs the response to experimental colitis in young adult rats." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 299, Issue 6, pp. G1376-85, (2010) (PubMed).

Target Details for BrdU

Target: BrdU (Bromodeoxyuridine (BrdU))

Alternative Name: BrdU (BrdU Products)

Target Type: Chemical

Background: Synonyms: 5-BROMODEOXYURIDINE