Anti-CD68 antibody [RM1031] - BSA and Azide free

• IHC

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the Kuffer cells in the human liver. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the macrophages in the human tonsil. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling CD68 with ab303565 at 1/100 (4.77 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 THP-1 cell lineNegative control : Jurkat (PMID : 17583472) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/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

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the macrophages in the mouse spleen. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, 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 ab303565 at 1/50 (9.54 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining in the red pulp of rat spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab303565 for 60mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.<\p> .

• IHC

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the macrophages in the rat spleen. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the Kuffer cells in the mouse liver. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC

Supplier Data

Immunohistochemistry - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling CD68 with ab303565 at 1/1000 (0.477 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positve staining on the Kuffer cells in the rat liver. The section was incubated with ab303565 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) was used. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of 100% cold methanol-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh) tissue labeling CD68 with ab303565 at 1/50 (9.54 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining in the red pulp of mouse spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab303565 for 60mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) cells labelling CD68 with ab303565 at 1/100 (4.77 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 is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/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.

• WB

Supplier Data

Western blot - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative controls : Jurkat, A20. The molecular mass observed is consistent with the literature (PMID : 7680921) Exposure time : 1 min

All lanes:

Western blot - Anti-CD68 antibody [RM1031] (<a href='/en-us/products/primary-antibodies/cd68-antibody-rm1031-ab303565'>ab303565</a>) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

A20 (mouse reticum sarcoma B lymphocyte) whole cell lysate at 20 µg

Lane 7:

NR8383 (rat alveolar macrophage) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 37 kDa

Observed band size: 110 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM /TBST Exposure time : Lane1 : 60 seconds; Lane2 : 5 seconds; Lane3 : 180 seconds; Lane4 : 7 seconds ab303565 works better than ab955 in western blot testing. We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results when using ab955 in western blot.

Lanes 1 and 3:

Western blot - Anti-CD68 antibody [KP1] (<a href='/en-us/products/primary-antibodies/cd68-antibody-kp1-ab955'>ab955</a>) at 1/1000 dilution

Lanes 2 and 4:

Western blot - Anti-CD68 antibody [RM1031] (<a href='/en-us/products/primary-antibodies/cd68-antibody-rm1031-ab303565'>ab303565</a>) at 1/1000 dilution

Lanes 1 - 2:

Human spleen tissue lysate at 20 µg

Lanes 3 - 4:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 37 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-CD68 antibody [RM1031] - BSA and Azide free (AB303566)

This data was developed using ab303565, the same antibody clone in a different buffer formulation. Western blot : Anti-CD68 antibody [RM1031] (ab303565) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab303565 was shown to bind specifically to CD68. A band was observed at 100 kDa in wild-type RAW 264.7 cell lysates with no signal observed at this size in CD68 knockout cell line. To generate this image, wild-type and CD68 knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CD68 antibody [RM1031] (<a href='/en-us/products/primary-antibodies/cd68-antibody-rm1031-ab303565'>ab303565</a>)

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

CD68 knockout RAW 264.7 cell lysate at 20 µg

Observed band size: 100 kDa

true