Anti-CD163 antibody [EPR19518]

29 product images

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  Flow Cytometry - Anti-CD163 antibody [EPR19518] (ab182422)

  Flow cytometry analysis of human PBMC cells (Human peripheral blood mononuclear cell) labeling with ab182422 at 1/60 dilution, 11.23 μg/ml (red). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) was used as the secondary antibody at 1/2000.

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  Image courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized human tonsil tissue stained for CD163 with ab182422 (30 mins at a 1/400 dilution) in immunohistochemical analysis. A Goat polyclonal HRP conjugate was used as the secondary.
  

  Heat mediated antigen retrieval buffer/enzyme used: pH 9.0 EDTA.

  Blocking step: 1% Protein Block ab64226 for 10 mins at RT.

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized mouse liver tissue stained for CD163 with ab182422 (45 mins at a 1/400 dilution) in immunohistochemical analysis. A Rabbit polyclonal HRP conjugate was used as the secondary.
  

  Heat mediated antigen retrieval buffer/enzyme used: pH 6.0 citrate.

  Blocking step: 1% Protein Block ab64226 for 10 mins at RT.

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized rat achilles tissue stained for CD163 with ab182422 (30 mins at a 1/200 dilution) in immunohistochemical analysis. A Rabbit polyclonal HRP conjugate was used as the secondary.
  

  Heat mediated antigen retrieval buffer/enzyme used: pH 6.0 citrate 70°C for 2hrs.

  Blocking step: 1% Protein Block ab64226 for 10 mins at RT.

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  Image courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  10% NBF, non-permeabilized mouse spleen tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Donkey anti Rabbit IgG polyclonal AlexaFluor^®647 conjugate was used as the secondary at a 1/200 dilution (red).
  

  Heat mediated antigen retrieval buffer/enzyme used: Sodium citrate pH 6.0.

  Blocking step: 5% serum for 1 hour at 21°C.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Cytoplasm staining on mouse spleen is observed. Counter stained with Hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Frozen sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse spleen tissue labeling CD163 with ab182422 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). The result showed some cytoplasmic staining on mouse spleen. The nuclear counterstain is DAPI (blue). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/1000 dilution. Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

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  Western blot - Anti-CD163 antibody [EPR19518] (ab182422)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  Exposure time: Lane 1: 1 minute; Lane 2-5: 3 minutes.

  U937 , THP-1 and J774A.1 cell lines were reported to be negative for CD163 expression.(PMID:16368951, 10648003 & 10577520).

  The molecular weight observed is consistent with what has been described in the literature (PMID:9712057 & 16517975).

  All lanes: Western blot - Anti-CD163 antibody [EPR19518] (ab182422) at 1/1000 dilution

  Lane 1: Human fetal liver lysate at 20 µg

  Lane 2: Human tonsil lysate at 20 µg

  Lane 3: Human fetal spleen lysate at 20 µg

  Lane 4: U937 (Human histiocytic lymphoma cell line) whole cell lysate at 20 µg

  Lane 5: THP-1 (Human monocytic leukemia cell line) whole cell lysate at 20 µg

  Lane 6: J774A.1 (Mouse macrophage reticulum cell sarcoma cell line) whole cell lysate at 20 µg

  Secondary

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

  Predicted band size: 125 kDa

  Observed band size: 150 kDa

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  10% NBF, non-permeabilized rat muscle tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Donkey anti Rabbit IgG polyclonal AlexaFluor^®647 conjugate was used as the secondary (red).
  

  Heat mediated antigen retrieval buffer/enzyme used: Tris/EDTA pH 9.0.

  Blocking step: 5% serum for 1 hour at 22°C.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Cytoplasm staining on Kupffer cells of human liver is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized human placenta tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Goat anti Rabbit polyclonal HRP conjugate was used as the secondary at a 1/200 dilution.
  

  Heat mediated antigen retrieval buffer/enzyme used: pH 6.0 citrate buffer.

  Blocking step: 3% serum for 30 mins at 20°C.

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  Western blot - Anti-CD163 antibody [EPR19518] (ab182422)

  Different batches of ab182422 were tested on Rat liver lysate at 2.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 150 kDa.

  All lanes: Western blot - Anti-CD163 antibody [EPR19518] (ab182422)

  Predicted band size: 125 kDa

• 

  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized mouse liver tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Goat anti Rabbit HRP conjugate was used as the secondary at a 1/200 dilution.
  

  Heat mediated antigen retrieval buffer/enzyme used: pH 6.0 citrate buffer.

  Blocking step: 3% serum for 30 mins at 20°C.

• 

  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  10% Formalin-fixed, non-permeabilized human breast carcinoma tissue stained for CD163 with ab182422 (12 hours, 4°C at a 1/200 dilution) in immunohistochemical analysis. A Donkey anti Rabbit IgG polyclonal AlexaFluor^®647 conjugate was used as the secondary at a 1/200 dilution (red).
  

  Heat mediated antigen retrieval buffer/enzyme used: Tris/EDTA pH 9.0.

  Blocking step: 5% serum for 1 hour at 22°C.

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Formaldehyde-fixed, non-permeabilized human first trimester placenta tissue stained for CD163 with ab182422 (16 hours, 4°C at a 1/250 dilution) in immunohistochemical analysis. A Pig anti Rabbit polyclonal biotin conjugate was used as the secondary at a 1/250 dilution.
  

  Heat mediated antigen retrieval buffer/enzyme used: Sodium citrate.

  Blocking step: 5% BSA for 30 mins at 22°C.

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  This image is courtesy of an anonymous customer review.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  10% NBF-fixed mouse spleen tissue stained for CD163 with ab182422 (18 hours at a 1/250 dilution) in immunohistochemical analysis. A Goat anti Rabbit IgG polyclonal AlexaFluor^®647 conjugate was used as the secondary at a 1/600 dilution (red).
  

  Heat mediated antigen retrieval buffer/enzyme used: Tris/EDTA pH 9.0.

  Blocking step: 20% serum for 1 hour at RT.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Cytoplasm staining on Hofbauer cells in human placenta is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Cytoplasm staining on Kupffer cells of mouse liver is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling CD163 with ab182422 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Cytoplasm staining on Kupffer cells of rat liver is observed. Counter stained with Hematoxylin.
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Frozen sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling CD163 with ab182422 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). The result showed some cytoplasmic staining on mouse liver. The nuclear counterstain is DAPI (blue).
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/1000 dilution. Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

• 

  Western blot - Anti-CD163 antibody [EPR19518] (ab182422)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  The molecular weight observed is consistent with what has been described in the literature (PMID:9712057 & 16517975).

  All lanes: Western blot - Anti-CD163 antibody [EPR19518] (ab182422) at 1/1000 dilution

  Lane 1: Mouse liver lysate at 10 µg

  Lane 2: Mouse heart lysate at 10 µg

  Lane 3: Mouse spleen lysate at 10 µg

  Lane 4: Mouse thymus lysate at 10 µg

  Lane 5: Rat liver lysate at 10 µg

  Lane 6: Rat heart lysate at 10 µg

  Lane 7: Rat spleen lysate at 10 µg

  Secondary

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

  Predicted band size: 125 kDa

  Observed band size: 150 kDa

  Exposure time: 3min

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  Multiplex immunohistochemistry - Anti-CD163 antibody [EPR19518] (ab182422)

  Fluorescence multiplex immunohistochemical analysis of human liver (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-MASP2 stained on cytoplasm of hepatocytes (Anti-MASP2 antibody [EPR23588-44] ab277520; gray; Opal™690) at 1:100 ( 5.22 μg/ml) [Panel B] , anti-CD163 stained on Kupffer cells (Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612; green; Opal™520) at 1:8000 ( 0.13 μg/ml) [Panel C], and anti-eNOS tained on endothelial cells (Anti-eNOS antibody [EPR23750-3] ab252439; red; Opal™570) at 1:200 ( 3.005 μg/ml) [Panel D] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. 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. The section was incubated in three rounds of staining: in the order of Anti-MASP2 antibody [EPR23588-44] ab277520, Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612, and Anti-eNOS antibody [EPR23750-3] ab252439 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.
  
  This data was developed using Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612, the same antibody clone in a different buffer formulation.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612).

  Immunohistochemical analysis of formalin fixed paraffin embedded Human tonsil labelling CD163 with Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612 anti CD163 antibody EPR19518 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Tissue Microarrays stained for "Anti-CD163 antibody [EPR19518]" using "ab182422"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab182422 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD163 with ab182422 at 2 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab182422 anti CD163 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD163 with ab182422 at 2 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab182422 anti CD163 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD163 antibody [EPR19518] (ab182422)

  Tissue Microarrays stained for "Anti-CD163 antibody [EPR19518]" using "ab182422"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab182422 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

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  Multiplex immunohistochemistry - Anti-CD163 antibody [EPR19518] (ab182422)

  Fluorescence multiplex immunohistochemical analysis of human liver (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-eNOS stained on endothelial cells (Anti-eNOS antibody [EPR23750-3] ab252439; red; Opal™570) at 1:1000 ( 1.004 μg/ml) [Panel B], anti-CD163 stained on Kupffer cells (Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612; green; Opal™520) at 1:8000 ( 0.13 μg/ml) [Panel B], and anti-Glucose Transporter GLUT2 stained on membrane of hepatocytes (Anti-Glucose Transporter GLUT2 antibody [EPR22946-74] ab234440; gray; Opal™690) at 1:200 ( 3.005 μg/ml) [Panel D] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. 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. The section was incubated in three rounds of staining: in the order of Anti-Glucose Transporter GLUT2 antibody [EPR22946-74] ab234440, Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612, and Anti-eNOS antibody [EPR23750-3] ab252439 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.
  
  This data was developed using Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612, the same antibody clone in a different buffer formulation.

• 

  Multiplex immunohistochemistry - Anti-CD163 antibody [EPR19518] (ab182422)

  This data was developed using Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612, the same antibody clone in a different buffer formulation.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver labelling GLUT2 with Anti-Glucose Transporter GLUT2 antibody [EPR22946-74] - BSA and Azide free ab260003 at 1/1000 (B), eNOS with Anti-eNOS antibody [RM1181] ab317582 at 1/500 dilution (C) and CD163 with Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612 at 1/8000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  
  Panel A: merged staining of anti-GLUT2 (magenta; Opal™690), anti-eNOS (green; Opal™520) and anti-CD163 (gray; Opal™570) on human liver.
  
  Panel B: anti-GLUT2 staining membrane of hepatocytes in human liver.
  
  Panel C: anti-eNOS staining endothelium in human liver.
  
  Panel D: anti-CD163 staining Kupffer cells in human liver.
  

  
  The section was incubated in three rounds of staining: in the order of Anti-Glucose Transporter GLUT2 antibody [EPR22946-74] - BSA and Azide free ab260003, Anti-eNOS antibody [RM1181] ab317582 and Anti-CD163 antibody [EPR19518] - BSA and Azide free ab213612 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.