Western blot - Calbindin antibody [AF2E5] (ab75524)
All lanes : Anti-Calbindin antibody [AF2E5] (ab75524) at 1/500 dilution
Lane 1 : Human kidney tissue lysate - total protein (ab30203) Lane 2 : Kidney (Rat) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 30 kDa Observed band size : 30 kDa
Exposure time : 90 seconds
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Calbindin antibody [AF2E5] (ab75524)This image is courtesy of an Abreview submitted by Karine Thibault
ab75524 staining rat brain (cortex) sections by IHC-FoFr. The animal was perfused with 4% paraformaldehyde and further post fixed with 4% paraformaldehyde overnight. The tissues were cryoprotected with 30% sucrose and sectioned using a cryostat. Staining with ab75524 at a 1/2800 dilution in PBS-Triton (0.3%) with 0.02% azide was performed for 18h at 25°C. A donkey anti-mouse Alexa488 polyclonal antibody at 1/1000 was used as the secondary antibody.
Overlay histogram showing SH-SY5Y cells stained with ab75524 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75524, 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 IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
ICC/IF image of ab75524 stained Hek293 cells. The cells were 4% PFA fixed (10mins) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75524, 1:1000 dilution) overnight at +4°C. The secondary antibody (green) was ab96879 Dylight 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.