Overlay histogram showing PC12 cells stained with ab225 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab225, 0.1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [B11/6](ab91353, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive result in 80% methanol (5 min) fixed PC12 cells used under the same conditions.

Immunocytochemistry/ Immunofluorescence analysis of horse adipose and bone marrow stem cells labeling CD90 / Thy1 with ab225 at 1/1000 dilution. Cells were fixed in paraformaldehyde and permeabilized with Triton x100 0.1%. The cells were blocked with 10% BSA for 30 minutes at 37°C, followed by staining with ab225 at 1/1000 for 12 hours in PBS+IGEPAL+BSA+10%NGS at 4°C. Goat F(ab')2 Anti-Mouse IgG - (Fab')2 (Biotin), pre-adsorbed (ab5886) was used as the secondary antibody at 1/400 dilution.

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Immunocytochemistry/ Immunofluorescence analysis of rat sciatic nerve schwann cells and fibroblasts labeling CD90 / Thy1 with ab225 at 1/400 dilution. Cells were fixed in paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were blocked with 5% serum for 1 hour at 21°C, followed by staining with ab225 at 1/400 in 0.2% BSA for 1 hour at 37°C. A polyclonal goat anti-mouse Alexa Fluor^® 546 secondary antibody was used at 1/500 dilution.

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All lanes : Anti-CD90 / Thy1 antibody [MRC OX-7] - Hematopoietic Stem Cell Marker (ab225) at 5 µg/ml

Lane 1 : Brain (Rat) Tissue Lysate
Lane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 17 kDa
Observed band size: 35-37 kDa why is the actual band size different from the predicted?


Exposure time: 20 minutes

Rat CD90/Thy1 is N-glycosylated at three sites, giving rise to molecules with a range of molecular masses (25-37 kDa). 

ab225 stained PC12 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab225 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

This image was kindly supplied as part of the review submitted by Nick Voilley. Rat retinal ganglion cell labelled with ab225 as a primary antibody and an anti-mouse F(ab)' 2 coupled to Alexa488 as a secondary antibody. The cell is approximately 15 micrometers in diameter. Only the cells labelled in green in the culture bear action potentials when stimulated. These three elements (reactivity to ab225, size and elecrophysiological parameters) clearly indicate the cell is a ganglion cell.