Immunohistochemical analysis of paraffin-embedded human breast tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human breast [PMID: 20103682].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed MDA-MB-231 (Human breast adenocarcinoma cell line) cell line labeling CD44 with ab189524 at 1/40 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

 

All lanes : Anti-CD44 antibody [EPR18668] (ab189524) 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: 82 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab189524 observed at 80 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab189524 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 ab189524 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDA-MB-231 (Human breast adenocarcinoma cell line) labeling CD44 with ab189524 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membranous staining on MDA-MB-231 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution.

 

All lanes : Anti-CD44 antibody [EPR18668] (ab189524) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000 dilution

Predicted band size: 82 kDa
Observed band size: 82 kDa


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

 

All lanes : Anti-CD44 antibody [EPR18668] (ab189524) at 1/2000 dilution

Lane 1 : Human thymus lysate
Lane 2 : Human skin lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000 dilution
Lanes 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 82 kDa
Observed band size: 82 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Due to high sequence homology between CD44 isoforms, within the immunogen region, this antibody cross reacts with most isoforms of CD44. The staining pattern is consistent with reference (PMID 19507218).

Lanes 1-6 : Anti-CD44 antibody [EPR18668] (ab189524) at 1/1000 dilution
Lanes 7-10 : Anti-CD44 antibody [EPR18668] (ab189524) at 1/20000 dilution

Lane 1 : Mouse brain lysate at 10 µg
Lane 2 : Mouse spleen lysate at 10 µg
Lane 3 : Rat brain lysate at 10 µg
Lane 4 : Rat heart lysate at 10 µg
Lane 5 : Rat kidney lysate at 10 µg
Lane 6 : Rat spleen lysate at 10 µg
Lane 7 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 8 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 9 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 10 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 82 kDa
Observed band size: 82 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1, 2, 3, 4, 5 and 6: 5 seconds; Lane 7, 8, 9 and 10: 1 second.

CD44v6 was immunoprecipitated from 1 mg of A549 (Human lung carcinoma cell line) whole cell lysate with ab189524 at 1/25 dilution. Western blot was performed from the immunoprecipitate using ab189524 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: A549 whole cell lysate 10 μg (Input).

Lane 2: ab189524 IP in A549 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab189524 in A549 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human kidney.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human tonsil.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human breast cancer [PMID: 15867228].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse colon.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse stomach.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse spleen.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on rat stomach.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on rat spleen.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.