Mouse eyes prepared for immunolabeling were fixed in 4% formalin and embedded in LR White acrylic resin (SPI, West Chester, PA). Thin sections (150 nm) were collected on 200-mesh nickel grids, dried for 24 h, and washed with a 1:1 dilution of blocker and PBS for 20 min (PBS, 0.1% Tween 20, 0.5% cold-water fish gelatin [Ted Pella, Inc., Redding, CA]), then a 1:50 dilution of rabbit anti-mouse histone H3 antibody (Abcam, Cambridge, UK; http://www.abcam.com/ ) in blocker solution for 1 h, washed in the blocker dilution once, soaked in a 1:100 dilution of gold-conjugated protein A (Sigma, St. Louis, MO; http://www.sigmaaldrich.com ) in blocker, then washed once in PBS and once in deionized water, then air-dried. Immunolabeled grids were stained with aqueous uranyl acetate (5%). In TEM micrographs, secondary gold-conjugated antibodies appear as small black dots with well-defined edges.
Gold conjugated protein a
Manufactured by Merck Group
Gold-conjugated protein A is a laboratory reagent used for the detection and purification of immunoglobulins. It consists of protein A, a bacterial cell wall protein that binds to the Fc region of antibodies, covalently attached to colloidal gold particles. This property allows for the visualization and capture of antibodies in various immunoassay and affinity chromatography applications.
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Lab products found in correlation
4 protocols using gold conjugated protein a
1
Immunolabeling Mouse Eye Sections
2
TCR Cluster Analysis by EM Imaging
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CCR5-/- and WT OT-II lymphoblasts treated - or not - with 2-DG (2 mM, 24 h) were used to prepare cell surface replicas as previously described (5 (link), 6 (link)). Briefly, T cells were fixed in 1% paraformaldehyde and labeled with anti-mouse CD3ϵ mAb (145-2C11), followed by 10 nm gold-conjugated protein A (Sigma-Aldrich). Labeled cells were adhered to poly-L-lysine-coated mica strips and fixed with 0.1% glutaraldehyde. Samples were covered with another mica strip, frozen in liquid ethane (KF-80, Leica), and stored in liquid nitrogen. Cell replicas were prepared with a Balzers 400T freeze fracture (FF) unit, mounted on copper grids, and analyzed using a JEM1010 electron microscope (Jeol) operating at 80 kV. Images were taken with a Bioscan CCD camera (Gatan) and processed with TVIPS software. EM image acquisition and quantification was performed by two researchers, one of them blind to the experiment. The number of TCR molecules in the same cluster was determined when the distance between gold particles was smaller than their diameter (10 nm).
3
Immunogold Labeling of T Cell Surface
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Immunogold‐labeled cell surface replicas were obtained as described (Kumar et al, 2011 ). Briefly, T cells were fixed in 1% paraformaldehyde (PFA) and labeled with anti‐mouse CD3 mAb (145‐2C11) or anti‐human CD3 mAb OKT3, followed by 10 nm gold‐conjugated protein A (Sigma‐Aldrich). Labeled cells were adhered to poly‐l ‐lysine‐coated mica strips and fixed with 0.1% glutaraldehyde. Samples were covered with another mica strip, frozen in liquid ethane (KF‐80, Leica), and stored in liquid nitrogen. Cell replicas were prepared with a Balzers 400T freeze fracture (FF) unit, mounted on copper grids, and analyzed on a JEM1010 electron microscope (Jeol, Japan) operating at 80 kV. Images were taken with a CCD camera (Bioscan, Gatan, Pleasanton, CA) and processed with TVIPS software (TVIPS, Gauting, DE). EM images were collected by two researchers, one of them blind to the experiment. Gold particles were counted on the computer. When distance between gold particles was smaller than their diameter (10 nm), they were considered part of the same cluster.
4
Immuno-gold Labeling of T Cell Surface
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Immunogold-labeled cell surface replicas were obtained as described (Kumar et al., 2011) .
Briefly, T cells were fixed in 1% paraformaldehyde (PFA) and labeled with anti-mouse CD3 mAb (145-2C11) or anti-human CD3 mAb OKT3, followed by 10 nm gold-conjugated protein A (Sigma-Aldrich). Labeled cells were adhered to poly-L-lysine-coated mica strips and fixed with 0.1% glutaraldehyde. Samples were covered with another mica strip, frozen in liquid ethane (KF-80, Leica), and stored in liquid nitrogen. Cell replicas were prepared with a Balzers 400T freeze fracture (FF) unit, mounted on copper grids, and analyzed on a JEM1010 electron microscope (Jeol, Japan) operating at 80 kV. Images were taken with a CCD camera (Bioscan, Gatan, Pleasanton, CA) and processed with TVIPS software (TVIPS, Gauting, DE). Gold particles were counted on the computer. When distance between gold particles was smaller than their diameter (10 nm), they were considered part of the same cluster.
Briefly, T cells were fixed in 1% paraformaldehyde (PFA) and labeled with anti-mouse CD3 mAb (145-2C11) or anti-human CD3 mAb OKT3, followed by 10 nm gold-conjugated protein A (Sigma-Aldrich). Labeled cells were adhered to poly-L-lysine-coated mica strips and fixed with 0.1% glutaraldehyde. Samples were covered with another mica strip, frozen in liquid ethane (KF-80, Leica), and stored in liquid nitrogen. Cell replicas were prepared with a Balzers 400T freeze fracture (FF) unit, mounted on copper grids, and analyzed on a JEM1010 electron microscope (Jeol, Japan) operating at 80 kV. Images were taken with a CCD camera (Bioscan, Gatan, Pleasanton, CA) and processed with TVIPS software (TVIPS, Gauting, DE). Gold particles were counted on the computer. When distance between gold particles was smaller than their diameter (10 nm), they were considered part of the same cluster.
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