Purified prion rods isolated from the equivalent of 0.5 ml of 10% (w/v) RML brain homogenate were labelled with anti-PrP monoclonal antibody SAF-32 (Bioquote Ltd, Cat # 189720) and goat anti-mouse IgG conjugated to 10 nm gold particles (Sigma-Aldrich, Cat # G7652) as described previously [10 (link)]. To label PrP rods in crude brain homogenate, an equal volume of 4% (w/v) sarkosyl was added to 100 µl 10% (w/v) RML brain homogenate followed by incubation at 37°C for 30 min. The sample was then centrifuged at 16 100g for 30 min. Pellets were resuspended in 100 µl of TBS containing 5% (v/v) glycerol and 0.1% (w/v) sarkosyl and passed through a 0.65 µm Ultrafree-MC Centrifugal PVDF filter unit (Millipore, Cat # UFC40DV25) to remove collagen fibres and intermediate filaments. Labelling of the filtrate fraction was then performed as described for purified prion samples [10 (link)].
Goat anti mouse igg conjugated to 10 nm gold particles
Manufactured by Merck Group
Goat anti-mouse IgG conjugated to 10 nm gold particles is a laboratory reagent used to detect and visualize mouse immunoglobulin G (IgG) in various applications, such as immunohistochemistry, Western blotting, and flow cytometry. The 10 nm gold particles serve as a contrast agent, allowing for the visualization of the target mouse IgG molecules.
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2 protocols using goat anti mouse igg conjugated to 10 nm gold particles
1
Prion Rod Labeling Protocol
2
Immunoelectron Microscopy of PDCoV
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Suspensions of purified PDCoV virions were placed onto nickel formvar-carbon coated grids (three grids per experiment) for approximately 10 min, followed by 1% bovine serum albumin (BSA) in 0.01 M PBS (pH 7.4) for 30 min. After, the samples were incubated with a mouse anti-NS6 antiserum for 30 min, or a pre-immune mouse serum as a negative control, with a rabbit anti-PDCoV hyperimmune serum and a rabbit anti-S1 antiserum as positive controls. All antibodies were diluted in 0.1% BSA and 0.05% Tween 20 in 0.01 M PBS (pH 7.4). The samples were then washed three times for 5 min per time with PBS and incubated with goat anti-mouse IgG conjugated to 10-nm gold particles (#G7652, Sigma) or goat anti-rabbit IgG conjugated to 10-nm gold particles (#G7402, Sigma). Grids were washed five times with PBS for 2 min per time, and then washed five times with Milli-Q water for 2 min per time, respectively. After the washing, samples were negatively stained with 3% uranyl acetate for 5 min and with lead citrate for 3 min and observed under electron microscopy.
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