Immunofluorescent analysis of 100% methanol-fixed HeLa cells labeling Ezrin with ab270442 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous and weak cytoplasmic staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

100% methanol fixation is recommended.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Ezrin with ab270442 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse kidney (PMID: 16522749). The section was incubated with ab270442 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized C6 (Rat glial tumor glial cell) cells labeling Ezrin with ab270442 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-Ezrin antibody [EPR23353-55] (ab270442) at 1/1000 dilution

Lane 1 : Mouse hippocampus tissue lysate
Lane 2 : Mouse fetal brain tissue lysate
Lane 3 : Rat brain tissue lysate
Lane 4 : Neuro-2a ( mouse neuroblastoma neuroblast) whole cell lysate
Lane 5 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 69 kDa

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

Exposure times: Lanes 1-6: 15 secs; Lane 7: 3 mins.

The band of 55kDa is the endogenously cleaved N-terminal form.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11124833, 17370041, 10388520).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ezrin with ab270442 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labeling Ezrin with ab270442 at 1/500 dilution (0.1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 100% methanol-fixed Neuro-2a cells labeling Ezrin with ab270442 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous and weak cytoplasmic staining in Neuro-2a cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

100% methanol fixation is recommended.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Ezrin with ab270442 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse spleen. The section was incubated with ab270442 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Ezrin with ab270442 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat kidney (PMID: 16522749). The section was incubated with ab270442 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Ezrin with ab270442 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on biliary epithelial cells in rat liver (PMID: 23507543). The section was incubated with ab270442 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Ezrin with ab270442 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat spleen.The section was incubated with ab270442 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Ezrin was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell) whole cell lysate with ab270442 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using 270442 1/1000 dilution (0.549 μg/ml). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C6 whole cell lysate 10 μg.
Lane 2: ab270442 IP in C6 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270442 in C6 whole cell lysate.

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

Exposure time: 84 secs.

The band of 55kDa is endogenously cleaved N-terminus form.

Ezrin was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab270442 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270442 at 1/1000 dilution (0.549 μg/ml). VeriBlot for IP Detection Reagent (HRP )(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg.

Lane 2: ab270442 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270442 in HeLa whole cell lysate.

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

Exposure time: 32 secs.

Ezrin was immunoprecipitated from 0.35 mg Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate with ab270442 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270442 at 1/1000 dilution (0.549 μg/ml). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Neuro-2a whole cell lysate 10 μg.
Lane 2: ab270442 IP in Neuro-2a whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270442 in Neuro-2a whole cell lysate.

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

Exposure time: 3 mins.

All lanes : Anti-Ezrin antibody [EPR23353-55] (ab270442) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : A549 ( human lung carcinoma epithelial cell) whole cell lysate
Lane 3 : MDA-MB-435S ( human mammary gland ductal carcinoma melanocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 69 kDa

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

Exposure time: 6 secs.

The band of 55kDa is the endogenously cleaved N-terminal form.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11124833, 17370041, 10388520).

Lower expression cell line: MDA-MB-435S (PMID: 17370041).

All lanes : Anti-Ezrin antibody [EPR23353-55] (ab270442) at 1/1000 dilution

Lane 1 : Human liver tissue lysate
Lane 2 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 69 kDa

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

Exposure time: 6 secs.

The band of 55kDa is the endogenously cleaved N-terminal form.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11124833, 17370041, 10388520).
Negative control: Human liver (PMID: 11124833).