Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247]

15 product images

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  Immunohistochemistry (Frozen sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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) (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) (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).

• 

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

  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 Anti-GAPDH antibody [EPR16891] - Loading Control 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 (Goat Anti-Rabbit IgG H&L (HRP) 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).

  All lanes: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

  Lane 1: HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg

  Lane 2: Wild-type HAP1 whole cell lysate at 40 µg

  Lane 3: Angiotensin Converting Enzyme 1 knockout HAP1 whole cell lysate at 40 µg

  Lane 4: Human kidney cell lysate at 40 µg

  Lane 5: Human lung cell lysate at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Predicted band size: 150 kDa

  Observed band size: 180 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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 (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 (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.

• 

  Immunohistochemistry (Frozen sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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) (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) (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).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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 (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 (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.

• 

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

  Blocking and dilution buffer: 5% NFDM/TBST.
  

  Exposure times.

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

  All lanes: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

  Lane 1: Rat brain tissue lysate at 20 µg

  Lane 2: Rat heart tissue lysate at 20 µg

  Lane 3: Rat liver tissue lysate at 20 µg

  Lane 4: Rat spleen tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 150 kDa

  Observed band size: 180 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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 (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 (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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222)

  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 (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 (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.

• 

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

  Blocking and dilution buffer: 5% NFDM/TBST.
  

  Exposure times.

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

  All lanes: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate at 20 µg

  Lane 2: Mouse heart tissue lysate at 20 µg

  Lane 3: Mouse kidney tissue lysate at 20 µg

  Lane 4: Mouse spleen tissue lysate at 20 µg

  Lane 5: Mouse lung tissue lysate at 20 µg

  Lane 6: bEND.3 (mouse brain endothelioma cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 150 kDa

  Observed band size: 180 kDa

• 

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

  ELISA analysis of ACE recombinant protein at 1000 ng/mL with ab254222. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

• 

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

  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 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423, ab254222, and Anti-Tissue Factor antibody [EPR22548-240] 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.

• 

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

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining GLEPP1/PTPRO with Anti-GLEPP1/PTPRO antibody [EPR28645-553] ab322047 at a 1:2000 (0.261 ug/ml) dilution, Anti-SLC12A3 antibody [EPR27106-48] ab316191 anti-SLC12A3 used at 1:2000 (0.254 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1:4000 (0.141 ug/ml) dilution.

  Panel A: merged staining of anti-PTPRO (magenta; Opal™690), anti-SCL12A3 (green; Opal™520) and anti-Angiotensin Converting Enzyme 1 (gray; Opal™570) on mouse kidney.
  
  Panel B: anti-PTPRO staining glomerulus in mouse kidney.
  
  Panel C: anti-SCL12A3 staining distal tubules in mouse kidney.
  
  Panel D: anti-Angiotensin Converting Enzyme 1 staining proximal tubules in mouse kidney.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GLEPP1/PTPRO antibody [EPR28645-553] ab322047, Anti-SLC12A3 antibody [EPR27106-48] ab316191 and ab254222 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.

  Nuclear counter stain with DAPI.

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

• 

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

  Fluorescence multiplex immunohistochemical analysis of the human kidney (Formalin/PFA-fixed paraffin-embedded sections).
  
  Panel A: merged staining of anti-Hexokinase 1 (Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423, green; Opal™690), anti-Angiotensin Converting Enzyme 1 (ab254222, gray; Opal™520) and anti-Aquaporin 2 (Anti-Aquaporin 2 antibody [EPR21080] 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 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423 at 1/250 dilution (4.224 μg/ml), ab254222 at 1/4000 dilution (0.141 μg/ml) and Anti-Aquaporin 2 antibody [EPR21080] 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.

• 

  Western blot - 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] (Anti-Calnexin antibody [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.

  Lanes 1 - 4: Western blot - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (ab254222) at 1/1000 dilution

  Lane 1: Wild-type SKNFI cell lysate at 20 µg

  Lane 2: Ace knockout SKNFI cell lysate at 20 µg

  Lane 3: Human Lung cell lysate at 20 µg

  Lane 4: HUVEC cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 150 kDa

  Observed band size: 200 kDa

• 

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

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining GLEPP1/PTPRO with Anti-GLEPP1/PTPRO antibody [EPR28645-553] ab322047 at a 1:2000 (0.261 ug/ml) dilution, Anti-SLC12A1/NKCC2 antibody [EPR28176-90] ab313640 anti-SLC12A1 used at 1:2000 (0.253 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1:4000 (0.141 ug/ml) dilution.

  Panel A: merged staining of anti-PTPRO (magenta; Opal™690), anti-SCL12A1 (green; Opal™520) and anti-Angiotensin Converting Enzyme 1 (gray; Opal™570) on mouse kidney.
  
  Panel B: anti-PTPRO staining glomerulus in mouse kidney.
  
  Panel C: anti-SCL12A1 staining distal tubules in mouse kidney.
  
  Panel D: anti-Angiotensin Converting Enzyme 1 staining proximal tubules in mouse kidney.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-GLEPP1/PTPRO antibody [EPR28645-553] ab322047, Anti-SLC12A1/NKCC2 antibody [EPR28176-90] ab313640 and ab254222 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.

  Nuclear counter stain with DAPI.

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