Anti-CD68 antibody [EPR23917-164]

22 product images

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  Flow Cytometry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling CD68 with ab283654 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, Goat F(ab')2 Anti-Rabbit IgG - H&L (DyLight® 488), pre-adsorbed ab98507) at 1/500 dilution was used as the secondary antibody.

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  Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
  

  Lanes 1-2: Merged signal (red and green). Green - ab283654 observed at 100 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
  ab283654 Anti-CD68 antibody [EPR23917-164] was shown to specifically react with CD68 in wild-type RAW264.7 cells. Loss of signal was observed when knockout cell line (knockout cell lysate - Mouse CD68 knockout RAW 264.7 cell lysate ab280106) was used. Wild-type and CD68 knockout samples were subjected to SDS-PAGE. ab283654 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654) at 1/1000 dilution

  Lane 1: Wild-type RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 2: Western blot - Mouse CD68 knockout RAW 264.7 cell lysate (Mouse CD68 knockout RAW 264.7 cell lysate ab280106) at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution

  Predicted band size: 37 kDa

  Observed band size: 100 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse spleen. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

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  Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (fresh) tissue labeling CD68 with ab283654 at 1/100 (4.66 ug/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution (Green). Positive staining on Kupffer cells of rat liver is observed. The nuclear counterstain was DAPI (Blue).
  

  Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

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  Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  Negative control: mouse skeletal muscle (PMID: 28091823).

  Exposure time: 37 seconds

  All lanes: Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654) at 1/1000 dilution

  Lane 1: Mouse spleen tissue lysate at 20 µg

  Lane 2: Mouse skeletal muscle tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

  Predicted band size: 37 kDa

  Observed band size: 100 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized J774A.1 cells labelling CD68 with ab283654 at 1/50 (9.32 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in J774A.1 cell line. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on immune cells in mouse colon. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat spleen (fresh) tissue labeling CD68 with ab283654 at 1/100 (4.66 ug/ml) dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution (Green). Positive staining on macrophage of rat spleen is observed. The nuclear counterstain was DAPI (Blue).
  

  Secondary antibody control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• 

  Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  Macrosialin (CD68) is a glycoprotein and can be de-glycosylated by PNGase F. The molecular mass observed is consistent with the literature (PMID: 7680921)

  Exposure time: Lane 1-2: 7.75 seconds; Lane 3-4: 48 seconds

  All lanes: Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654) at 1/1000 dilution

  Lane 1: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 15 µg

  Lane 2: RAW264.7 treated with PNGase F whole cell lysate at 15 µg

  Lane 3: Mouse spleen tissue lysate at 15 µg

  Lane 4: Mouse spleen treated with PNGase F tissue lysate at 15 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 37 kDa

  Observed band size: 100 kDa

• 

  Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  Exposure time: Lane 1: 7.75 seconds; Lane 2: 125 seconds; Lane 3: 92 seconds

  All lanes: Western blot - Anti-CD68 antibody [EPR23917-164] (ab283654) at 1/1000 dilution

  Lane 1: J774A.1 (mouse reticum cell sarcoma monocyte macrophage) whole cell lysate 20 at 20 µg

  Lane 2: Rat liver tissue lysate at 20 µg

  Lane 3: Rat spleen tissue lysate at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

  Predicted band size: 37 kDa

  Observed band size: 100 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Mouse large B cell lymphoma tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse large B cell lymphoma. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) .

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat spleen. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Rat lung tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on macrophages in rat lung. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of paraffin-embedded Rat liver tissue labelling CD68 with ab283654 at 1/100 (4.66 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Kupffer cells in rat liver. The section was incubated with ab283654 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse colon tissue staining CD68 with ab283654 at a 1/100 dilution, Anti-CD19 antibody [EPR23174-145] ab245235 anti-CD19 used at 1/1000 dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  Panel A: merged staining of anti-CD68 (magenta; Opal^™520), anti-CD19 (green; Opal^™690) and anti-CD3 (grey; Opal^™570) on mouse colon.
  
  Panel B: anti-CD68 staining macrophages in mouse colon.
  
  Panel C: anti-CD19 staining B lymphocytes in mouse colon.
  
  Panel D: anti-CD3 staining T lymphocytes in mouse colon.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of ab283654, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.

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  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining CD68 with ab283654 at a 1/100 dilution, Anti-CD19 antibody [EPR23174-145] ab245235 anti-CD19 used at 1/1000 dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  Panel A: merged staining of anti-CD68 (magenta; Opal^™520), anti-CD19 (green; Opal^™690) and anti-CD3 (grey; Opal^™570) on mouse small intestine.
  
  Panel B: anti-CD68 staining macrophages in mouse small intestine.
  
  Panel C: anti-CD19 staining B lymphocytes in mouse small intestine.
  
  Panel D: anti-CD3 staining T lymphocytes in mouse small intestine.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of ab283654, Anti-CD19 antibody [EPR23174-145] ab245235 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.

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  Immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Immunohistochemical analysis of formalin fixed paraffin embedded mouse spleen labelling CD68 with ab283654 at a concentration of 0.1µ/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.

  ab283654 anti-CD68 antibody [EPR23917-164] 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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  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  CD68 Multiplex immunohistochemistry staining of mouse lung tissue using rabbit Anti-CD68 antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse lung tissue staining MARCO with Anti-MARCO antibody [RM1219] ab323717 at a 1:200 (2.49 ug/ml) dilution, ab283654 anti-CD68 used at 1:100 (4.43 ug/ml) dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1:2000 (0.26 ug/ml) dilution.

  Panel A: merged staining of anti-MARCO (green; Opal™520), anti-CD68 (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on mouse lung.
  
  Panel B: anti-MARCO staining macrophages in mouse lung.
  
  Panel C: ant-CD68 staining macrophages in mouse lung.
  
  Panel D: ant-CD3 staining T lymphocytes in mouse lung.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-MARCO antibody [RM1219] ab323717, ab283654 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• 

  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  CD68 Multiplex immunohistochemistry staining of rat liver tissue using rabbit Anti-CD68 antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat liver tissue staining MARCO with Anti-MARCO antibody [RM1219] ab323717 at a 1:200 (2.49 ug/ml) dilution, ab283654 anti-CD68 used at 1:100 (4.43 ug/ml) dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1:2000 (0.26 ug/ml) dilution.

  Panel A: merged staining of anti-MARCO (green; Opal™520), anti-CD68 (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on rat liver.
  
  Panel B: anti-MARCO staining macrophages in rat liver.
  
  Panel C: ant-CD68 staining macrophages in rat liver.
  
  Panel D: ant-CD3 staining T lymphocytes in rat liver.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-MARCO antibody [RM1219] ab323717, ab283654 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• 

  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  CD68 Multiplex immunohistochemistry staining of Mouse liver using rabbit Anti-CD68 antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse liver tissue staining CD5L/CT-2 with Anti-CD5L/CT-2 antibody [EPR29364-21] ab324193 at a 1:500 (0.958 ug/ml) dilution, ab283654 anti-CD68 used at 1:100 (4.43 ug/ml) dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1:2000 (0.25 ug/ml) dilution.

  Panel A: merged staining of anti-CD5 antigen-like (green; Opal™520), anti-CD68 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on mouse liver.
  
  Panel B: anti-CD5 antigen-like staining macrophages in mouse liver.
  
  Panel C: anti-CD68 staining macrophages in mouse liver.
  
  Panel D: anti-CD3 staining T lymphocytes in mouse liver.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-CD5L/CT-2 antibody [EPR29364-21] ab324193, ab283654 and Anti-CD3 epsilon antibody [CAL57] ab237721 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• 

  Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] (ab283654)

  CD68 Multiplex immunohistochemistry staining of human liver tissue using rabbit Anti-CD68 antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human liver tissue staining MARCO with Anti-MARCO antibody [RM1219] ab323717 at a 1:200 (2.49 ug/ml) dilution, ab283654 anti-CD68 used at 1:500 (1.26 ug/ml) dilution and Anti-CD3 epsilon antibody [CAL57] ab237721 anti-CD3 used at a 1:2000 (0.26 ug/ml) dilution.

  Panel A: merged staining of anti-MARCO (green; Opal™520), anti-CD68 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on human liver.
  
  Panel B: anti-MARCO staining macrophages in human liver.
  
  Panel C: ant-CD68 staining macrophages in human liver.
  
  Panel D: ant-CD3 staining T lymphocytes in human liver.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-MARCO antibody [RM1219] ab323717, Anti-CD68 antibody [EPR20545] ab213363 and Anti-CD3 epsilon antibody [CAL57] ab237721 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.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• 

  Flow Cytometry (Intracellular) - Anti-CD68 antibody [EPR23917-164] (ab283654)

  Flow cytometry staining of C57BL/6 mouse splenocytes (top) or C57BL/6 Mouse Bone Marrow Cell-derived Macrophages (bottom) with ab283654 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. Cells were incubated for 30min on ice in 1x PBS containing 10µg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab283654 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶in 100 µl at 1.0 µg/ml (1/2212)) for 30min at 22°C. The cells were simultaneously stained with F4/80.

  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

  Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.