重组Anti-CD10抗体[EPR22865-73] (ab255609)

Fluorescence multiplex immunohistochemical analysis of the human endometrium (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-E Cadherin (ab256580, red; Opal™690), anti-SLC34A2 (ab238793, green; Opal™520) and anti-CD10 (ab255609, cyan; Opal™570) on human endometrium. Panel B: anti-CD10 stained on stromal cells. Panel C: anti-E Cadherin stained on glandular cells. Panel D: anti-SLC34A2 stained on apical membrane of glandular cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab256580 at 1/3000 dilution  (0.324 μg/ml) for 30mins, ab238793 at 1/1000 dilution (2.26 μg/ml) for 10mins and ab255609 at 1/1000 dilution (0.615 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

Fluorescence multiplex immunohistochemical analysis of the human breast (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-B7H4 (ab252438, red; Opal™690), anti-CD10 (ab255609, gray; Opal™520) and anti-FABP4 (ab92501, cyan; Opal™570) on human breast. Panel B: anti-B7H4 stained on glandular lumens. Panel C: anti-CD10 stained on myoepithelial cells. Panel D: anti-FABP4 stained on adipocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab252438 at 1/100 dilution (4.69 μg/ml), ab255609 at 1/1000 dilution (0.615 μg/ml) and ab92501 at 1/10000 dilution (0.047 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

All lanes : Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution

Lane 1 : Human placenta tissue lysate
Lane 2 : Raji (human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : Ramos (human Burkitt's lymphoma cell line) whole cell lysate
Lane 4 : HT-29 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 5 : Wild-type HAP1 whole cell lysate
Lane 6 : CD10 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution


Predicted band size: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

ab255609 was shown to specifically react with CD10 in wild-type HAP1 cells as signal was lost in CD10 knockout cells. Wild-type and CD10 knockout samples were subjected to SDS-PAGE. ab255609 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

Lanes 5-6 in this blot were developed using a higher sensitivity ECL substrate.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 10 seconds; Lanes 2-4: 15 seconds; Lanes 5-6: 70 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

Negative control: HT-29 (PMID:19828468).

 

CD10 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab255609 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255609 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: Raji whole cell lysate 10 μg (Input).
Lane 2: ab255609 IP in Raji whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255609 in Raji whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Immunofluorescent analysis of 100% methanol-fixed Ramos (human Burkitt's lymphoma cell line) cells labeling CD10 with ab255609 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining in Ramos cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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

100% methanol was preferred as fixative.

Immunofluorescent analysis of 100% methanol-fixed Raji (human Burkitt's lymphoma cell line) cells labeling CD10 with ab255609 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining in Raji cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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

100% methanol was preferred as fixative.

Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution + Mouse kidney tissue lysate at 20 µg

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

Predicted band size: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

 

Immunohistochemical analysis of paraffin-embedded Human diffuse large B-cell lymphoma labelling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP polymer) ready to use (ab214880). Positive staining on human diffuse large B-cell lymphoma is observed. Counter stained with hematoxylin.

 

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

 

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution

Lane 1 : Rat kidney tissue lysate
Lane 2 : Rat liver tissue 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: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on germinal center of human tonsil (PMID:10843287) is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

 

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on intrahepatic canaliculi of human liver (PMID:10705818) is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on myoepithelial cells of human breast (PMID:10705818, 17143263) is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on proximal convoluted tubules and glomerular epithelial cells of human kidney (PMID:10705818) is observed. Counter stained with hematoxylin.

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

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).