Recombinant Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

False colour image of Western blot: Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab254222 was shown to bind specifically to Angiotensin Converting Enzyme 1. A band was observed at 200 kDa in wild-type SKNFI cell lysates with no signal observed at this size in Ace knockout cell line ab288707. To generate this image, wild-type and Ace knockout SKNFI 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.

Fluorescence multiplex immunohistochemical analysis of the human kidney (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-Hexokinase 1 (ab150423, green; Opal™690), anti-Angiotensin Converting Enzyme 1 (ab254222, gray; Opal™520) and anti-Aquaporin 2 (ab199975, red; Opal™570) on human kidney. Panel B: anti-Aquaporin 2 stained on collecting tubules. Panel C: anti-Angiotensin Converting Enzyme 1 stained on proximal tubules. Panel D: anti-Hexokinase 1 stained on distal tubules and collecting tubules. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab150423 at 1/250 dilution (4.224 μg/ml), ab254222 at 1/4000 dilution (0.141 μg/ml) and ab199975 at 1/4000 dilution (0.152 μg/ml) for 30 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.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

Fluorescence multiplex immunohistochemical analysis of paraffin-embedded Human kidney tissue.

Panel A: Merged staining of anti-Hexokinase 1 (gray; Opal™690), anti-Angiotensin Converting Enzyme 1 (green; Opal™520) and anti-Tissue Factor (red; Opal™570) on human kidney.
Panel B: Anti-Tissue Factor stained on renal glomeruli.
Panel C: Anti-Angiotensin Converting Enzyme 1 stained on proximal tubules.
Panel D: Anti-Hexokinase 1 stained on distal tubules.

The section was incubated in three rounds of staining: in the order of ab150423, ab254222, and ab252918 for 30 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. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Counterstained with DAPI.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized rat kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Positive staining on proximal tubules of kidney (PMID:10504496) is observed. The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lanes 1-3: 3 minutes. Lanes 4-5: 5.5 seconds.

ab254222 was shown to specifically react with Angiotensin Converting Enzyme 1 in wild-type HAP1 cells as signal was lost in Angiotensin Converting Enzyme 1 knockout cells. Wild-type and Angiotensin Converting Enzyme 1 knockout samples were subjected to SDS-PAGE. ab254222 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

The molecular weight observed, and the expression profile are consistent with what have been described in the literature (PMID: 25495544, 16203874).

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules of mouse kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab254222 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of frozen section of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Positive staining on proximal tubules of kidney (PMID:10504496) is observed. The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules in mouse kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 48 seconds. Lanes 2 & 3:  3 minutes. Lane 4: 48 seconds.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on blood vessels of human liver (PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human lkidney tissue labeling Angiotensin Converting Enzyme 1 with ab254222 at 1/4000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on proximal tubules of human kidney (PMID: 2828286; PMID: 175444) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is 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.

The section was incubated with ab254222 for 30 mins at room temperature. 
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lanes 1-4: 10 seconds. Lane 5: 5.5 seconds. Lane 6: 48 seconds.