Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human esophageal carcinoma tissue sections labeling CD44 with Purified ab101531 at 1/100 dilution (1.03 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-CD44 antibody [SP37] (ab101531) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CD44 knockout HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : LnCAP cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 80 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab101531 observed at 80 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab101531 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab101531 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Formalin-fixed, paraffin-embedded human esophageal carcinoma tissue stained for CD44 with ab101531 at a 1/100 dilution in immunohistochemical analysis.

Anti-CD44 antibody [SP37] (ab101531) at 1/25 dilution + HeLa cell lysate

Predicted band size: 80 kDa
Observed band size: 80 kDa