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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SU-DHL-1 cells labelling CD163 with ab182422 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 µg/mL) (Green). BM:Confocal image showing membranous staining in SU-DHL-1 cell line ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution (2 µg/mL).

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).

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

Lane 1 : Human fetal liver lysate
Lane 2 : Human tonsil lysate
Lane 3 : Human fetal spleen lysate
Lane 4 : U937 (Human histiocytic lymphoma cell line) whole cell lysate
Lane 5 : THP-1 (Human monocytic leukemia cell line) whole cell lysate
Lane 6 : J774A.1 (Mouse macrophage reticulum cell sarcoma cell line) 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: 121 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

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).

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

Lane 1 : Mouse liver lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Mouse thymus lysate
Lane 5 : Rat liver lysate
Lane 6 : Rat heart lysate
Lane 7 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 121 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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