Recombinant Anti-CD163 antibody [EPR19518] (ab182422)

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Red; TG700N), anti-CD163 (ab182422; Brown; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Violet; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain. The inset image shows the separate CD163 signal.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Violet; TG700N), anti-CD163 (ab182422; Red; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Orange; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

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.

See Abreview

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, ab150081) was used as the secondary antibody at 1/2000.

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) (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 ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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) (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 ab150077 at 1/1000 dilution. Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

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).

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.

See Abreview

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% ab64226 for 10 mins at RT.

See Abreview

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.

See Abreview

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.

See Abreview

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.

See Abreview

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.

See Abreview

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.

See Abreview

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% ab64226 for 10 mins at RT.

See Abreview

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.

See Abreview

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) (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 ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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) (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 ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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) (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 ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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) (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 ab97051 at 1/500 dilution. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 

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) (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 ab150077 at 1/1000 dilution. Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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 (ab209101). The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

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 (ab209101). The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.