Anti-CLC3 antibody [N258/5] (ab134285) at 1/200 dilution + Rat brain membrane lysate at 15 µg

Secondary
Goat Anti-Mouse IgG: HRP at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 91 kDa

4% formaldehyde-fixed SK-N-BE cells stained for CLC3 (green) using ab134285 at 1/100 dilution in ICC/IF.

Secondary Antibody: Goat Anti-Mouse ATTO 488 at 1/100 for 60 minutes at room temperature. Counterstain: Phalloidin Texas Red F-Actin stain; DAPI (blue) nuclear stain at 1/1000, 1/5000 for 60 minutes room temperature, 5 minutes room temperature.

(A) DAPI (blue) nuclear stain. (B) Phalloidin Texas Red F-Actin stain. (C) ab134285. (D) Composite.

IHC image of CLC3 staining in human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab134285, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Overlay histogram showing SH-SY5Y cells stained with ab134285 (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 (ab134285, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.