重组Anti-CYP11A1抗体[EPR24868-86] (ab272494)

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. 

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. 

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. anti-DDX4 / MVH stained on all spermatogenic cell types.

Panel C: anti-DDX4 / MVH stained on all spermatogenic cell types.

Panel D: anti-Vimentin stained on Sertoli cells and fibroblasts.

Key protocol steps: 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

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 time: Lane 1: 7.75 seconds Lane 2-4: 37 seconds

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 (BNOD™, Polymer Refine Detection) was used. 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) was used.

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

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) was used.

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

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 is observed. 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).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

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.

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 10µg

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

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 time: Lane 1-4, 7-9: 3 minutes Lane 5-6: 37 seconds

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) was used. 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) was used.

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

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.

CYP11A1 was immunoprecipitated from 0.35 mg Mouse testis tissue lysate with ab272494 at 1/30 dilution (2 μg 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 10µg

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

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)was used. 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) was used.

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

CYP11A1 was immunoprecipitated from 0.35 mg Rat testis tissue lysate with ab272494 at 1/30 dilution (2 μg 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 10µg

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

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)was used.

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