Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labelling CYP11A1 with ab272494 at 1/10000 (0.054 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human cardiac muscle. The section was incubated with ab272494 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labelling CYP11A1 with ab272494 at 1/10000 (0.054 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Leydig cells of human testis. The section was incubated with ab272494 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

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

Fluorescence multiplex immunohistochemical analysis of human adrenal gland (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-PCSK2 stained on adrenal medulla (ab274418; gray; Opal™690) at 1 : 2000 (0.263 μg/ml) [Panel B], anti-CYP11A1 stained on adrenal cortex (ab272494; green; Opal™520) at 1 : 10000 (0.053 μg/ml) [Panel C], and anti-Neuropeptide Y stained on chromaffin cells (ab221145; red; Opal™570) at 1 : 2000 (0.279 μg/ml) [Panel D] on human adrenal gland. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary antibody. 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. The section was incubated in three rounds of staining : in the order of ab274418, ab272494, and ab221145 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.  This data was developed using ab272494, the same antibody clone in a different buffer formulation.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human adrenal gland tissue labelling CYP11A1 with ab272494 at 1/10000 (0.054 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human adrenal cortex (PMID : 22796438). (A) Low‑powered (magnification, x40) and (B) high‑powered (magnification, x400) microscopic images. The section was incubated with ab272494 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

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

Fluorescence multiplex immunohistochemical analysis of human adrenal gland (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-PCSK2 stained on adrenal medulla (ab274418; gray; Opal™690) at 1 : 2000 (0.263 μg/ml) [Panel B], anti-CYP11A1 stained on adrenal cortex (ab272494; green; Opal™520) at 1 : 10000 (0.053 μg/ml) [Panel C], and anti-Collagen VI stained on extracellular matrix (ab182744; red; Opal™570) at 1 : 1000 (1.518 μg/ml) [Panel D] on human adrenal gland. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary antibody. 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. The section was incubated in three rounds of staining : in the order of ab274418, ab272494, and ab182744 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.  This data was developed using ab272494, the same antibody clone in a different buffer formulation.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue.

Panel A : merged staining of anti-Vimentin (red; Opal™690), anti-CYP11A1 (cyan; Opal™520) and anti-DDX4 / MVH (green; Opal™570) on human testis.
Panel B : anti-CYP11A1 stained on Leydig cells.
Panel C : anti-DDX4 / MVH stained on all spermatogenic cell types.
Panel D : anti-Vimentin stained on Sertoli cells and fibroblasts.

The section was incubated in three rounds of staining : in the order of ab193555 (1 : 2000 dilution) and ab272494 (1 : 10000 dilution) for 30 mins, then ab270534 (1 : 2000 dlilution) for 10 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.

DAPI was used as a nuclear counter stain. Opal Polymer HRP Ms + Rb was used as a secondary.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized JEG-3 (Human placenta epithelial choriocarcinoma) cells labelling CYP11A1 with ab272494 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 Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized JEG-3 (human placenta epithelial choriocarcinoma) cells labelling CYP11A1 with ab272494 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining colocalized with mitochondrial marker (ab33985) in JEG-3 cell line. ab33985 Anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain mitochondrion at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab272494 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution.

-ve control 2 : ab33985 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

CYP11A1 was immunoprecipitated from 0.35 mg JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate with ab272494 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272494 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate 10 ug

Lane 2 : ab272494 IP in JEG-3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab272494 in JEG-3 whole cell lysate

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

Exposure time : 58 seconds

All lanes:

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] (<a href='/en-us/products/primary-antibodies/cyp11a1-antibody-epr24868-86-ab272494'>ab272494</a>)

Predicted band size: 60 kDa

Observed band size: 50 kDa

false

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab313783).

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat adrenal gland staining of VGF with ab313783 at a 1/500 (0.96 µg/ml) dilution, CYP11A1 with ab272494 at a 1/10000 (0.053 µg/ml) dilution and Neuropeptide Y with ab221145 at a 1/4000 (0.140 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.

Panel A : merged staining of anti-VGF (green; Opal™690), anti-CYP11A1 (magenta; Opal™520) and anti-Neuropeptide Y (gray; Opal™570) on rat adrenal gland.
Panel B : anti-VGF staining endocrine cells in rat adrenal gland medulla.
Panel C : anti-CYP11A1 staining adrenal cortex in rat adrenal gland.
Panel D : anti-Neuropeptide Y staining chromaffin cellsa in rat adrenal gland medulla.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab313783, ab272494 and ab221145 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat adrenal gland tissue labelling CYP11A1 with ab272494 at 1/10000 (0.054 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat adrenal cortex. The section was incubated with ab272494 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling CYP11A1 with ab272494 at 1/10000 (0.054 ug/ml) followed by a ready to use LeicaDS9800(Bond™ Polymer Refine Detection). Positive staining on Leydig cells of mouse testis. The section was incubated with ab272494 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 (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-12 (Rat adrenal gland pheochromocytoma) cells labelling CYP11A1 with ab272494 at 1/50 dilution (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 Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab272494).

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse adrenal gland staining of VGF with ab313783 at a 1/500 (0.96 µg/ml) dilution, CYP11A1 with ab272494 at a 1/10000 (0.053 µg/ml) dilution and Neuropeptide Y with ab221145 at a 1/4000 (0.140 µg/ml) dilution followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.

Panel A : merged staining of anti-VGF (green; Opal™690), anti-CYP11A1 (magenta; Opal™520) and anti-Neuropeptide Y (gray; Opal™570) on mouse adrenal gland.
Panel B : anti-VGF staining endocrine cells in mouse adrenal gland medulla.
Panel C : anti-CYP11A1 staining adrenal cortex in mouse adrenal gland.
Panel D : anti-Neuropeptide Y staining endocrine cells in mouse adrenal gland medulla.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab313783, ab272494 and ab221145 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

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

• IP

Lab

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

CYP11A1 was immunoprecipitated from 0.35 mg Rat testis tissue lysate with ab272494 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272494 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Rat testis tissue lysate 10 ug

Lane 2 : ab272494 IP in Rat testis tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab272494 in rat testis tissue lysate

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

Exposure time : 58 seconds

All lanes:

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] (<a href='/en-us/products/primary-antibodies/cyp11a1-antibody-epr24868-86-ab272494'>ab272494</a>)

Predicted band size: 60 kDa

Observed band size: 50 kDa

false

• IP

Lab

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

CYP11A1 was immunoprecipitated from 0.35 mg Mouse testis tissue lysate with ab272494 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272494 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse testis tissue lysate 10 ug

Lane 2 : ab272494 IP in Mouse testis tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab272494 in mouse testis tissue lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-CYP11A1 antibody [EPR24868-86] (<a href='/en-us/products/primary-antibodies/cyp11a1-antibody-epr24868-86-ab272494'>ab272494</a>)

Predicted band size: 60 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

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

Negative control : human heart (PMID : 19491374, 21520051).

The band below (~37 kDa) in lane 2 may be caused by degradation.

Exposure times : Lane 1 : 7.75 seconds; Lane 2-4 : 37 seconds.

All lanes:

Western blot - Anti-CYP11A1 antibody [EPR24868-86] (<a href='/en-us/products/primary-antibodies/cyp11a1-antibody-epr24868-86-ab272494'>ab272494</a>) at 1/1000 dilution

Lane 1:

Human adrenal gland tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human heart tissue lysate at 20 µg

Lane 4:

JEG-3 (Human placenta epithelial choriocarcinoma) 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: 60 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-CYP11A1 antibody [EPR24868-86] - BSA and Azide free (AB288573)

This data was developed using ab272494, the same antibody clone in a different buffer formulation.

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

Low expression : brain (PMID : 21520051, 19491374).

Lane 7-9 : This blot was developed using a higher sensitivity ECL substrate.

The band below (~37 kDa) in lane 1 may be caused by degradation.

Exposure times : Lane 1-4, 7-9 : 3 minutes; Lane 5-6 : 37 seconds

All lanes:

Western blot - Anti-CYP11A1 antibody [EPR24868-86] (<a href='/en-us/products/primary-antibodies/cyp11a1-antibody-epr24868-86-ab272494'>ab272494</a>) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat testis tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Mouse ovary tissue lysate at 20 µg

Lane 6:

Rat ovary tissue lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 9:

Rat-1 (rat embryonic fibroblast) whole cell 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 1/100000 dilution

Predicted band size: 60 kDa

Observed band size: 50 kDa

false