All lanes : Anti-alpha Internexin antibody (ab7259) at 1/10000 dilution

Lane 1 : Protein standard
Lane 2 : Mouse spinal cord lysate
Lane 3 : Rat spinal cord lysate
Lane 4 : Cow spinal cord lysate

Predicted band size: 66 kDa

Immunofluorescence analysis of rat cerebellum section stained for alpha Internexin using ab7259 at a 1/2000 dilution (green) co-strained with chicken polyclonal antibody to GFAP at a 1/5000 dilution (red). DAPI was used to stain nuclear DNA (blue).

Following transcardial perfusion with 4% paraformaldehyde, brain was post-fixed for 24 hours, cut to 45 μM, and free-floating sections were stained.

Ab7259 shows red, neuronal progenitor cells, plectin (not one of our antibodies) shows fibroblast marker. Photo courtesy of: Dr. Gerry Shaw University of Florida

 

Ab7259 staining Human normal temporal cortex. Staining is localised to intracellualr compartment.
Left panel: with primary antibody diluted 1:2000. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ICC/IF image of ab7259 stained PC12 cells. The cells were 100% methanol fixed (5 min) 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 (ab7259, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.