IdU antibody

Catalog No. ABIN2669973

Product Details anti-IdU Antibody

Target: IdU (Iododeoxyuridine (IdU))

Reactivity: Chemical

Host: Mouse

Clonality: Monoclonal

Conjugate: This IdU antibody is un-conjugated

Application: Immunohistochemistry (IHC), Immunofluorescence (IF)

Purification: Purified from mouse ascites fluids by affinity chromatography

Immunogen: Iododeoxyuridine coupled to keyhole limpet hemocyanin.

Clone: OTI2B10

Isotype: IgG2b

Application Details

Application Notes: IHC 1:150, IF 1:150,

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #103750 (Immunohistochemistry)

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

No.: #103750

Date: 04/19/2019

Antigen: IdU

Lot Number: W001

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. ABIN2669973 works in IHC-P with a microwave antigen retrieval pretreatment and concentration of 1:50.

Validation Images

Staining of mouse brain and intestine sections using ABIN2669973. A-B: A bright staining in the nuclei of individual cells scattered across the brain is present, especially in the sub-cortical areas at the border between the gray and white matters (A) and ventricular wall (B). Abbreviations: GM = gray matter; LV = lateral ventricle; WM = white matter. C-E: positive control staining in the intestine (duodenum). IdU+ proliferating cells are clearly detected in the germinal layer of the intestinal epithelium, at the basis of the duodenal villi.

Full Methods

Primary Antibody: ABIN2669973

Secondary Antibody: goat anti-mouse AF594-conjugated (Invitrogen by Thermo Fisher Scientific, A11032, lot 819561)

Full Protocol:

• Inject mouse intraperitoneally at the dose of 0.057mg/g body weight with IdU (Sigma, I7125) dissolved in sterile distilled water at final concentration of 100mg/ml, pH7.5.
• Perfuse mouse with 4% paraformaldehyde in 0.1M phosphate buffer (PB) pH7.4 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-IdU (antibodies-online, ABIN2669973, lot W001) diluted 1:50 in PBS-BSA-PLL ON at 4°C.
• Wash sections 3x 5min with 0.01M PBS.
• Incubate sections with secondary goat anti-mouse AF594-conjugated (Invitrogen by Thermo Fisher Scientific, A11032, lot 819561) diluted 1:500 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 ABIN2669973 and 1:20, 1:70, and 1:100 dilutions were also tested with 0.01M PBS, 5% Normal Goat Serum (NGS Sigma, G9023, lot SLBV1396) and 0.1% Triton-X -100 as the blocking solution, but 1:50 and PBS containing 1% albumin from chicken egg white and 0.3% Triton-X gave the best results.

Handling

Format: Liquid

Concentration: 4.54 mg/mL

Target Details for IdU

Target: IdU (Iododeoxyuridine (IdU))

Alternative Name: IdU (IdU Products)