Anti-CD68 antibody [EPR23917-164] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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 (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, ab98507) at 1/500 dilution was used as the secondary antibody.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining CD68 with ab283654 at a 1/100 dilution, ab245235 anti-CD19 used at 1/1000 dilution and 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, ab245235 and 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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse colon tissue staining CD68 with ab283654 at a 1/100 dilution, ab245235 anti-CD19 used at 1/1000 dilution and 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, ab245235 and 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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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

• WB

Lab

Western blot - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/cd68-antibody-epr23917-164-ab283654'>ab283654</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 37 kDa

Observed band size: 100 kDa

false

• WB

Supplier Data

Western blot - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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 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 - ab280106) was used. Wild-type and CD68 knockout samples were subjected to SDS-PAGE. ab283654 and 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 (ab216773) and 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] (<a href='/en-us/products/primary-antibodies/cd68-antibody-epr23917-164-ab283654'>ab283654</a>) 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 (<a href='/en-us/products/cell-lysates/mouse-cd68-knockout-raw-2647-cell-lysate-ab280106'>ab280106</a>) at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 37 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/cd68-antibody-epr23917-164-ab283654'>ab283654</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 37 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-CD68 antibody [EPR23917-164] - BSA and Azide free (AB283667)

This data was developed using ab283654, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/cd68-antibody-epr23917-164-ab283654'>ab283654</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 37 kDa

Observed band size: 100 kDa

false