This is a knockout-validated antibody summary, based on three publications "Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: Implications for visual manifestations of Parkinson's disease" [3], "A novel resource for studying function and dysfunction of α-synuclein: mouse lines for modulation of endogenous Snca gene expression" [2], "α-Synuclein deficiency promotes neuroinflammation by increasing Th1 cell-mediated immune responses" [1], "Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders" for (figure 2a, 2b1) [4], and "Knocking out alpha-synuclein in melanoma cells dysregulates cellular iron metabolism and suppresses tumor growth" for western blot knockout validation (figure 2a) [5]. Article "Sensitivity and specificity of phospho-Ser129 α-synuclein monoclonal antibodies" also indicates the knockout validation of this antibody (see figure 7 of the article) [6]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Company: BD transduction

Antibody: α-synuclein (α-syn)

Catalog number: 610786

Summary: Mouse monoclonal IgG1 raised against rat synuclein-1 aa. 15-123. Reactivity: Rat (QC Testing) Human, Mouse (Tested in Development). Application: Western blot (Routinely Tested), Immunofluorescence (Tested During Development), Immunohistochemistry (Reported).

IHC, immunohistochemistry

Enucleated eyes fixed in 4% paraformaldehyde in PBS overnight followed by 95% ethanol for a minimum of 24 h at room temperature.

Fixed eyes were embedded in paraffin, and 4 μm sections were immuno-stained using the standard protocol.

For DAB staining, sections were treated with 3% H2O2 in PBS (pH 11.95) for 60 min to bleach melanin.

Antigen retrieval was performed by heating in the presence of 10mM citric buffer (pH 6) in a pressure cooker set in a microwave oven for 10min.

For immunostaining with fluorescent secondary antibodies, sections were deparaffinized and rehydrated sections before being subjected to antigen retrieval by heating to 97oC in the presence of 25mM tris-1mM EDTA (pH 8.5) for 40 min. Sections were then blocked in 1% BSA.

See Fig 1. in [3]. Retinal sections from α-syn+/+ mice show positive immunoreaction for α-syn in all retinal cell layers. Sections from α-syn-/- mice show no reactivity for α-syn as expected.

Samples were lyzed in RIPA lysis buffer (50 mM Tris-Cl pH7.4, 100 mM NaCl, 1% NP-40, 0.5% deoxycholate).

Dilutions of α-syn antibody for immunoblotting was 1:2000.

See Fig 2 in [3]. Retinal, brain, spleen, liver lysates were examined.

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Figure 1. Western blot analysis reveals the presence of α-synuclein (α-syn) in antigen-presenting cells (CD11b+ and CD11c+) and CD4+ T cells isolated from the spleen of aSyn+/+ mice. Spinal cord (SC) lysates of aSyn+/+ and aSyn−/− mice were loaded as positive and negative controls for α-synuclein immunoreactivity. From [1].

Western blot (Figure 1).

Spinal cord (SC) lysates from aSyn+/+ and aSyn−/− mice.

1:300 dilution.

1:1,000 dilution donkey anti-mouse 488 fluorescence-conjugated antibody (Dianova).

Signal was detected with Fusion FX7 detection system (PeqLab).

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Figure 2. Western blot analysis of total protein samples extracted from the cerebral cortex (Cx) and midbrain (Mb) of a homozygous (SNCAfloxΔneo/floxΔneo) α-synuclein floxed mouse after deletion of the neo-cassette and two homozygous α-synuclein floxed mice after deletion of the exon II by Cre/loxP recombination (SNCAΔflox/Δflox). Beta-actin was used as loading control. From [2].

Western blot (Figure 2).

Brain tissues of homozygous α-synuclein floxed mice with and without Cre/loxP recombination. Mouse tissues were homogenized in the SDS-PAGE loading buffer following incubation for 10 min at 100 C.

1:1,000 dilution.

HRP-conjugated antibodies.

Chemiluminescent-based detection.