This is a knockout-validated antibody summary, based on the publication "Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Cell lysates from HEK-293 parental, heterozygous (+/-) or two individual C9ORF72 KO clones (KOA, KOB) were prepared and processed for immunoblot with the indicated C9ORF72 antibodies. The arrows point to positive C9ORF72 signals. The Ponceau stained transfers associated with each blot are shown as a protein loading control. lysis of 16 C9ORF72 antibodies by immunoblot.

Mouse monoclonal IgG2a

Company: GeneTex

Antibody: C9orf72

Catalog number: GTX634482

Summary: Mouse monoclonal IgG2a against a recombinant protein encompassing a sequence within the N-terminus region of human C9orf72. Reacts with mouse and human. Suitable for western blot and immunohistochemistry (paraffin).

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Figure 2. Longer exposure immunoblots on HEK-293 cell lysates and immunoblots of brain lysates from WT and C9ORF72 KO mice with the indicated C9ORF72 antibodies. The arrows point to positive C9ORF72 signals.

Western blot | Immunohistochemistry

HEK-293 parental, heterozygous (+/-) and C9ORF72 KO clones, and WT and C9ORF72 KO brain tissues.

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Figure 3. DAB immunohistochemistry of C9ORF72 WT and C9ORF72 KO mouse brains in the sagittal plane using C9ORF72 antibody GTX634482. Micrographs are ordered from the rostral to caudal aspect of the mouse brain. For C9ORF72 WT, arrows indicate areas of increased DAB labeling intensity and correspond to the inset regions in the middle column of images. For C9ORF72 KO (right most column), arrows indicate areas of non-specific labeling. Scale bars = 100 um or 25 um for the inset images on the right for the C9ORF72 WT micrographs). From [1].

WB: Cultured cells were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 5% glycerol, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor.

IHC: Formalin-fixed, paraffin-embedded brain tissue from 3 month old C9-KO (n = 3) and C9-WT (n = 3) mice were sectioned, deparaffinized and heat-induced epitope retrieval was performed.

WB: 5% milk in TBS with 0.1% Tween 20 (TBST).

IHC: 2.5% normal horse serum and 0.3% Triton X-100 in TBS for 1 hr at room temperature.

WB: 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST) O/N at 4˚C.

IHC: diluted in DAKO antibody diluent (Agilent, Cat# S0809) at 1:5,000 and incubated O/N at 4°C.

WB: 1:10,000 dilution anti-rabbit (Jackson ImmunoResearch Laboratories) in TBST with 5% milk for 1 hr at room temperature.

IHC: ImmPRESS HRP horse anti-mouse IgG (Vector Labs, Cat# MP-7402), for 1 hr at room temperature.

WB: Detection of immuno-reactive bands was performed by image scan using a LI-COR Odyssey Imaging System (LI-COR Biosciences).

IHC: ImmPACT DAB peroxidase substrate kit (Vector Labs, Cat# SK-4105). Slides were then counterstained with Hematoxylin Solution, Gill No.1 (Sigma-Aldrich, Cat# GHS132) for 5 min at room temperature. Micrographs were captured with a digital camera mounted on a Leica DM6000 B upright microscope with either 10X or 40X objectives using Volocity Imaging software (version 6.3.0 PerkinElmer).

The antibody is not very specific for ICC, but it is the best for IHC. The signal is nearly completely ablated in the KO mice although there is some non-specific signal seen in the dentate gyrus/CA4, inferior olive and cerebellar cortex sections.