Anti-CD68 antibody [KP1] - BSA and Azide free

• Mass Cytometry

Collaborator

Mass Cytometry - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

Imaging Mass Cytometry™ (IMC™) image of human tonsil tissue stained with Anti-CD68 antibody [KP1]. ab233172 (carrier-free antibody, purified) was metal-conjugated using a Maxpar® Antibody Labeling Kit from Fluidigm. Immunostaining was performed according to Fluidigm's protocols. Briefly, slides were subject to deparaffinization and heat-induced epitope retrieval, followed by overnight incubation at 4°C with an antibody cocktail containing metal-tagged antibodies in blocking buffer. Slides were subsequently washed with 0.2% Triton-X and 1x PBS, counterstained with Cell-ID™ Intercalator-Ir diluted at 1/400 in 1x PBS for 30 min at room temperature, rinsed for 5 min with distilled H2O, and air-dried prior to IMC™ acquisition. IMC™ acquisition was performed using the Fluidigm Hyperion™ Imaging System.

Imaging Mass Cytometry™, IMC™, Cell-ID™, Hyperion™ and Maxpar® are trademarks of Fluidigm Canada

This image is courtesy of the Single Cell & Imaging Mass Cytometry Analysis Platform, Goodman Cancer Research Centre, McGill University

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD68 with ab233172 at 1/3000 dilution, followed by Goat Anti-mouse IgG H&L (HRP) ready to use. Cytoplasmic staining on macrophages of human tonsil (PMID : 19543531) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-mouse IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

Immunofluorescent analysis of 100% methanol-fixed THP-1 (human monocytic leukemia cell line) cells labeling CD68 with ab233172 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with Recombinant Anti-beta IV Tubulin antibody [EPR16775] (ab179504) at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/1000 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue labelling CD68 with ab955 at 1/3000 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab93684 a Goat Anti-mouse IgG H&L (HRP) was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Cytoplasmic staining on Kupffer cells of human liver (PMID : 12118106).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab955).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

This data was produced using the same antibody clone but in a different formulation containing PBS, sodium azide, glycerol and BSA (ab955).

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling CD68 with ab955 at a concentration of 0.1 µg/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.

ab955 Anti-CD68 antibody [KP1] 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)

• WB

Supplier Data

Western blot - Anti-CD68 antibody [KP1] - BSA and Azide free (AB233172)

This data was developed using the same antibody clone in a different buffer formulation (ab955). Blocking anf diluting buffer and concentraion : 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

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