Anti-CD33 antibody [EPR23051-101]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunohistochemical analysis of formalin fixed paraffin embedded human hodgkins lymphoma labelling CD33 with ab269456 at a concentration of 1µg/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.

ab269456 anti-CD33 antibody [EPR23051-101] 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)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control：Human skeletal muscle.

The section was incubated with ab269456 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on human Hodgkin's lymphoma.

The section was incubated with ab269456 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on mainly Kuffer cells of human liver (PMID : 25721896). The section was incubated with ab269456 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunofluorescent analysis of 100% methanol-fixed THP-1 (human monocytic leukemia monocyte) and Jurkat (human T cell leukemia T lymphocyte) cells labeling CD33 with ab269456 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Confocal image showing membranous staining in THP-1 cells. The nuclear counter stain is DAPI (Blue). Tubilin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red).

Control : Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Negative control : Jurkat (PMID : 30519686).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD33 antibody [EPR23051-101] (AB269456)

Immunohistochemical analysis of paraffin-embedded Human clear cell renal cell carcinoma tissue labeling CD33 with ab269456 at 1/200 dilution (2.96 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Membranous staining on infiltrated myeloid cells of human clear cell renal cell carcinoma The section was incubated with ab269456 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IP

Unknown

Immunoprecipitation - Anti-CD33 antibody [EPR23051-101] (AB269456)

CD33 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate with ab269456 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269456 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : THP-1 whole cell lysate 10ug.

Lane 2 : ab269456 IP in THP-1 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab269456 in THP-1 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 23 seconds.

All lanes:

Immunoprecipitation - Anti-CD33 antibody [EPR23051-101] (ab269456)

Predicted band size: 40 kDa

Observed band size: 67-75 kDa

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• WB

Supplier Data

Western blot - Anti-CD33 antibody [EPR23051-101] (AB269456)

False colour image of Western blot : Anti-CD33 antibody [EPR23051-101] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab269456 was shown to bind specifically to CD33. A band was observed at 60-80 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CD33 knockout cell line ab273831 (knockout cell lysate ab273785). To generate this image, wild-type and CD33 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-CD33 antibody [EPR23051-101] (ab269456) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 10 µg

Lane 2:

Western blot - Human CD33 knockout THP-1 cell lysate (ab273785) at 10 µg

Lane 3:

HL-60 cell lysate at 10 µg

Lane 4:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 60-80 kDa

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• WB

Unknown

Western blot - Anti-CD33 antibody [EPR23051-101] (AB269456)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 70 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16380601, 30519686).

Negative control：Human skeletal muscle.

All lanes:

Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 40 kDa

Observed band size: 75 kDa

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• WB

Unknown

Western blot - Anti-CD33 antibody [EPR23051-101] (AB269456)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 70 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16380601, 30519686).

Negative Control：Jurkat (PMID : 30519686).

All lanes:

Western blot - Anti-CD33 antibody [EPR23051-101] (ab269456) at 1/1000 dilution

Lane 1:

U937 (human histiocytic lymphoma monocyte), whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

Jurkat (human T cell leukemia T lymphocyte), 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: 40 kDa

Observed band size: 67-75 kDa

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