Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free

19 product images

• 

  Flow Cytometry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  This data was developed using the same antibody clone in a different buffer formulation (ab225894)
  

  Flow cytometry overlay histogram showing wild-type A431 (green line) and EPCAM knockout A431 cells stained with Anti-EpCAM antibody [EPR20532-225] ab223582 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (Anti-EpCAM antibody [EPR20532-225] ab223582) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 4°C.

  The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used at 1/2000 for 30 min at 4°C.

  Isotype control antibody was Rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) used at the same concentration and conditions as the primary antibody (wild-type A431 - black line; EPCAM knockout A431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• 

  Western blot - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).
  

  Lanes 1 - 3: Merged signal (red and green). Green - Anti-EpCAM antibody [EPR20532-225] ab223582 observed at 40 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

  Anti-EpCAM antibody [EPR20532-225] ab223582 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with Anti-EpCAM antibody [EPR20532-225] ab223582 and Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-EpCAM antibody [EPR20532-225] (Anti-EpCAM antibody [EPR20532-225] ab223582) at 1/1000 dilution

  Lane 1: Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: EPCAM knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: Western blot - Human EPCAM knockout A-431 cell line (Human EPCAM knockout A-431 cell line ab261902)

  Lane 3: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 35 kDa

  Observed band size: 40 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Immunohistochemical analysis of paraffin-embedded human thyroid cancer tissue labeling EpCAM with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human thyroid cancer is observed (PMID: 15637741). 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EpCAM antibody [EPR20532-225] ab223582).

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

• 

  Flow Cytometry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Clone EPR20532-225 (ab225894) has been successfully conjugated by Abcam. This image was generated using Anti-EpCAM antibody [EPR20532-225] (PE). Please refer to PE Anti-EpCAM antibody [EPR20532-225] ab237397 for protocol details.

  Overlay histogram showing HCT116 cells stained with PE Anti-EpCAM antibody [EPR20532-225] ab237397 (red line). The cells were then incubated in 1x PBS / 10% normal Goat serum to block non-specific protein-protein interactions followed by the antibody (PE Anti-EpCAM antibody [EPR20532-225] ab237397, 1/6250 dilution) for 30 min at 4°C. Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (PE Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582). Anti-EpCAM antibody [EPR20532-225] ab223582 staining EpCAM in T47D positive cells (top panel) and HeLa negative cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-EpCAM antibody [EPR20532-225] ab223582 at 0.1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Immunofluorescent analysis of 4% paraformaledehyde-fixed, 0.1% Triton X-100 permeabilized HT-29 (human colorectal adenocarcinoma cell line) cells labeling EpCAM with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining on HT-29 cells.

  The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889) (red) at 1/200 dilution.

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

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EpCAM antibody [EPR20532-225] ab223582).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Immunohistochemical analysis of paraffin-embedded human colon tissue labeling EpCAM with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human colon is observed (PMID: 15637741). 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EpCAM antibody [EPR20532-225] ab223582).

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

• 

  Immunoprecipitation - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  EpCAM was immunoprecipitated from 0.35 mg of HCT 116 (human colorectal carcinoma cell line) whole cell lysate with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10000 dilution.
  

  Lane 1: HCT 116 lysate 10 μg (Input).

  Lane 2: Anti-EpCAM antibody [EPR20532-225] ab223582 IP in HCT 116 lysate.

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-EpCAM antibody [EPR20532-225] ab223582 in HCT 116 whole cell lysate.

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

  Exposure time: 30 seconds.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EpCAM antibody [EPR20532-225] ab223582).

  All lanes: Immunoprecipitation - Anti-EpCAM antibody [EPR20532-225] (Anti-EpCAM antibody [EPR20532-225] ab223582)

  Predicted band size: 35 kDa

  Observed band size: 42 kDa

  Exposure time: 30s

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582). Anti-EpCAM antibody [EPR20532-225] ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-EpCAM antibody [EPR20532-225] ab223582 at 0.1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582). Anti-EpCAM antibody [EPR20532-225] ab223582 staining EpCAM in wild-type A431 cells (top panel) and EpCAM knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/5000 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
  Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Clone EPR20532-225 (ab225894) has been successfully conjugated by Abcam. This image was generated using Anti-EpCAM antibody [EPR20532-225] (Alexa Fluor® 647). Please refer to Alexa Fluor® 647 Anti-EpCAM antibody [EPR20532-225] ab237396 for protocol details.

  Alexa Fluor® 647 Anti-EpCAM antibody [EPR20532-225] ab237396 staining EpCAM in HT-29 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with Alexa Fluor® 647 Anti-EpCAM antibody [EPR20532-225] ab237396 at 1/100 dilution (shown in red) and Alexa Fluor® 488 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in ). Nuclear DNA was labeled with DAPI (shown in blue).

  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  This product also gave a positive signal under the same testing conditions in HT-29 cells fixed with 100% methanol (5 min).

• 

  Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Clone EPR20532-225 (ab225894) has been successfully conjugated by Abcam. This image was generated using Anti-EpCAM antibody [EPR20532-225] (Alexa Fluor® 488). Please refer to Alexa Fluor® 488 Anti-EpCAM antibody [EPR20532-225] ab237395 for protocol details.

  Alexa Fluor® 488 Anti-EpCAM antibody [EPR20532-225] ab237395 staining EpCAM in HT-29 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with Alexa Fluor® 488 Anti-EpCAM antibody [EPR20532-225] ab237395 at 1/1000 dilution (shown in green) and Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab202272, Rabbit monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labeled with DAPI (shown in blue).

  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of the human endometrium (Formalin/PFA-fixed paraffin-embedded sections).
  
  Panel A: merged staining of anti-EpCAM (Anti-EpCAM antibody [EPR20532-225] ab223582, magenta; Opal™690), anti-NKG2A (Anti-NKG2A antibody [EPR23737-127] ab260035, green; Opal™520) and anti-CD31 (Anti-CD31 antibody [EPR17259] ab182981, red; Opal™570) on human endometrium. Panel B: anti-NKG2A stained on NK cells. Panel C: anti-CD31 stained on endothelial cells. Panel D: anti-EpCAM stained on 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 Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/500 dilution (1.008 μg/ml), Anti-NKG2A antibody [EPR23737-127] ab260035 at 1/2000 dilution (0.262 μg/ml) and Anti-CD31 antibody [EPR17259] ab182981 at 1/4000 dilution (0.137 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.
  
  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).

  This data is courtesy of ImmunoAtlas and it can be found here.

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).

• 

  This image is courtesy of ImmunoAtlas.

  Multiplex immunohistochemistry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Anti-PD-L1 (Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611; cyan; Opal™ 520), Anti-Granzyme B (Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803; yellow; Opal™ 540), Anti-PD1 (Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596; green; Opal™ 650) and Anti-FOXP3 (Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

  The immunostaining was performed on a Leica Biosystems BOND® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

  The section was incubated in six rounds of staining; sequentially for Anti-PD-L1 antibody [CAL10] - BSA and Azide free ab251611 (1/750 dilution), Anti-Granzyme B antibody [EPR20129-217] - BSA and Azide free ab219803 (1/250 dilution), Anti-PD1 antibody [CAL20] - BSA and Azide free ab251613 (1/750 dilution), Anti-pan Cytokeratin antibody [C-11] - BSA and Azide free ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), Anti-CD8 alpha antibody [CAL66] - BSA and Azide free ab251596 (1/1500 dilution) and Anti-FOXP3 antibody [236A/E7] - BSA and Azide free ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

  This data is courtesy of ImmunoAtlas and it can be found here.

  
  This data was developed using the same antibody clone in a different buffer formulation (Anti-EpCAM antibody [EPR20532-225] ab223582).
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  Flow Cytometry - Anti-EpCAM antibody [EPR20532-225] - BSA and Azide free (ab225894)

  Flow cytometric analysis of HCT 116 (human colorectal carcinoma cell line) cell line labeling EpCAM with Anti-EpCAM antibody [EPR20532-225] ab223582 at 1/500 (red) compared with a rabbit monoclonal IgG Isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), at 1/2000 dilution was used as the secondary antibody.

  Total viable cells were gated for the final data.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-EpCAM antibody [EPR20532-225] ab223582).