NeuN antibody (AA 1-140)

Catalog No. ABIN5542704

Product Details anti-NeuN Antibody

Target: NeuN (RBFOX3) (RNA Binding Protein, Fox-1 Homolog 3 (RBFOX3))

Binding Specificity: AA 1-140

Reactivity: Human, Mouse

Host: Mouse

Clonality: Monoclonal

Conjugate: This NeuN antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), ELISA, Immunocytochemistry (ICC), Flow Cytometry (FACS)

Immunogen: Purified recombinant fragment of human RBFOX3 (AA 1-140) expressed in E. Coli.

Clone: 1H3B2

Isotype: IgG2a

Application Details

Application Notes: ELISA: 1:10000, WB: 1:500 - 1:2000, IHC: , ICC: 1:200 - 1:1000, FCM: 1:200 - 1:400

Restrictions: For Research Use only

Independent Validation (WB)

Validation #104496 (Western Blotting)

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

No.: #104496

Date: 08/02/2023

Antigen: RBFOX3

Lot Number: 160425

Method validated: Western Blotting

Positive Control:

Adult mouse brain and cerebellum

Notes:

The NeuN antibody ABIN5542704 works in WB at a 1:1000 dilution with sensitive ECL substrate on adult mouse brain and cerebellum extracts. Non-specific bands are also detected.

Validation Images

Western blot using ABIN5542704 to reveal CALB1 (28 kDa) in extracts from two different mouse brains (1 and 2) and in cerebellum extracts (3).

Full Methods

Primary Antibody: ABIN5542704

Secondary Antibody:

HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, NA931VS)

Full Protocol:

• Homogenize tissues with cold lysis buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1% protease inhibitor (Sigma P8340) using an ultrasonic homogenizer (MSE, SoniPrep 150) with 16 amplitude, 20 s on, 10 s off pulse for 60 s.
• Centrifuge tissue homogenates at 13,000 rpm for 20 min at 4 °C.
• Collect supernatants and determine total protein content using a Bradford assay.
• Denature 50 µg of total protein for 5 min at 90 °C and subsequently separate them on a denaturing 12% PAGE-SDS gel alongside a Precision Plus Protein Dual Color Standard (Bio-Rad, 160374).
• Electro-transfer proteins onto nitrocellulose membrane (Amersham Biosciences, RPN203D) ON in the cold room.
• Wash membrane 3x for 10 min with 0.01 M PBS containing 0.1% Tween-20 (PBST).
• Block membrane with PBST containing 2% bovine serum albumin for 1 h at RT.
• Incubate membrane with primary mouse anti-NeuN antibody (antibodies-online, ABIN5542704, lot 160425) diluted 1:1,000 in PBST ON at 4 °C.
• Wash membrane 3x 10 min with PBST.
• Incubate membrane with secondary HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, NA931VS) diluted 1:1,000 in PBST for 1 h at RT.
• Wash membrane 3x 10 min with PBST.
• Visualize proteins with WesternBright Sirius HRP substrate (Advansta) using a ChemiDoc Imaging System.

Experimental Notes:

Non-specific bands were also detected in adult mouse cerebellum tissue extracts, but not on laser-microdissected Purkinje neurons.

Independent Validation (IHC)

Validation #104533 (Immunohistochemistry)

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

No.: #104533

Date: 01/26/2024

Antigen: NeuN

Lot Number: 160425

Method validated: Immunohistochemistry

Positive Control:

Adult (> 2 months) CD1 mouse brain fixed in 4% paraformaldehyde.

Negative Control:

One control slice for each experimental procedure processed omitting the primary antibody; overnight incubation in diluent solution only.

Notes:

Passed. NeuN antibody (AA 1-140) ABIN5542704 works in for IHC-P at 1:100 concentration with microwave antigen retrieval.

Validation Images

Neu-N immunoreactive neuronal nuclei in the mouse cerebral cortex. The area in the rectangle is shown at higher magnification in the right panel. Original magnification 20x – insert 40x.

Full Methods

Primary Antibody: ABIN5542704

Secondary Antibody: anti-rabbit secondary antibody Alexa Fluor 594 (Thermo Fisher A11037, lot # 2439060)

Full Protocol:

• Perfuse mice with paraformaldehyde 4% in 0.1 M phosphate buffer pH 7.4 and post-fix of samples in the same fixative for an additional 2 h at RT.
• Wash, dehydrate, and embed samples in paraffin wax.
• Wash several times with 0.01 M PBS.
• Wash several times with 0.01 M PBS.
• Cut brain with a microtome into 6 µm sections and mount on glass slides.
• After paraffin removal sections were processed for microwave antigen retrieval for 10 minutes (95-100° C) in 10 mM sodium citrate buffer (pH 6.0). After 20 minutes of spontaneous cooling, sections were washed (distilled water, 2 times for 5 minutes each; followed by PBS for 5 minutes).
• Incubate sections for 1 h at RT in a humid chamber in PBS containing 1% ovalbumin (Sigma, A5378) and 0.3% Triton-X-100 (BioRad, 161-0407, lot 00583) to block non-specific binding sites.
• Incubate sections with primary rabbit NeuN antibody (AA 1-140) (antibodies-online, ABIN5542704, lot 160425) diluted 1:50 and 1:100 in PBS-BSA-PLL (Sigma, P1524) ON at RT in a humid chamber.
• Wash sections 3x 5 min with 0.01 M PBS.
• Incubate sections with anti-rabbit secondary antibody Alexa Fluor 594, 1:500, in 0.1M PBS (Thermo Fisher A11037, lot # 2439060) for 1 h at RT.
• Wash sections 3x 5 min with 0.01 M PBS.
• Specimens were mounted in Fluoroshield (Sigma, F6182, lot # MKCB0153V.
• Images were acquired with a Leica DM 6000B fluorescence microscope with a digital camera at 40x magnification.

Handling

Format: Liquid

Buffer: PBS with 0.05 % sodium azide

Storage: 4 °C,-20 °C

Storage Comment: 4°C, -20°C for long term storage

Target Details for NeuN

Target: NeuN (RBFOX3) (RNA Binding Protein, Fox-1 Homolog 3 (RBFOX3))

Alternative Name: RBFOX3 (RBFOX3 Products)

Synonyms: FOX-3 antibody, FOX3 antibody, HRNBP3 antibody, NEUN antibody, Fox-3 antibody, Hrnbp3 antibody, NeuN antibody, Neuna60 antibody, Neun antibody, RGD1560070 antibody, RNA binding fox-1 homolog 3 antibody, RNA binding protein, fox-1 homolog (C. elegans) 3 antibody, RNA binding protein, fox-1 homolog 3 antibody, RBFOX3 antibody, Rbfox3 antibody

Background: RBFOX3 (RNA Binding Protein, Fox-1 Homolog (C. Elegans) 3) is a Protein Coding gene. Diseases associated with RBFOX3 include vertebral artery occlusion and tuberculum sellae meningioma. Among its related pathways are Neuroscience. GO annotations related to this gene include nucleic acid binding and nucleotide binding. An important paralog of this gene is RBFOX1.,

Molecular Weight: 28.3kDa

HGNC: 23492