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

• IHC-P

Unknown

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 (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.

• IHC-P

Unknown

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 (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.

• mIHC

Lab

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 (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.

• mIHC

Lab

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 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.

• IHC-P

Unknown

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 (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.

• IHC-P

Unknown

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 (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.

• mIHC

Supplier Data

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 ab322047 at a 1 : 2000 (0.261 ug/ml) dilution, 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 ab322047, 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (AB254222)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat kidney tissue staining NADPH oxidase 4 with ab319055 at a 1 : 500 (1.032 ug/ml) dilution, ab233837 anti-Hexokinase 1 used at 1 : 250 (4.36 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1 : 4000 (0.132 ug/ml) dilution.

Panel A : merged staining of anti-NADPH oxidase 4 (green; Opal™520), anti-Hexokinase 1 (magenta; Opal™690) and anti-Angiotensin Converting Enzyme 1 (yellow; Opal™570) on rat kidney.
Panel B : anti-NADPH oxidase 4 staining distal tubules in rat kidney.
Panel C : anti-Hexokinase 1 staining distal tubules in rat kidney.
Panel D : anti-Angiotensin Converting Enzyme 1 staining proximal tubules in rat kidney.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab319055, ab233837 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (AB254222)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining NADPH oxidase 4 with ab319055 at a 1 : 500 (1.032 ug/ml) dilution, ab199975 anti-Aquaporin 2 used at 1 : 4000 (0.143 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1 : 4000 (0.132 ug/ml) dilution.

Panel A : merged staining of anti-NADPH oxidase 4 (green; Opal™520), anti-Aquaporin 2 (magenta; Opal™690) and anti-Angiotensin Converting Enzyme 1 (yellow; Opal™570) on mouse kidney.
Panel B : anti-NADPH oxidase 4 staining distal tubules in mouse kidney.
Panel C : anti-Aquaporin 2 staining collecting 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 ab319055, ab199975 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (AB254222)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat kidney tissue staining NADPH oxidase 4 with ab319055 at a 1 : 500 (1.032 ug/ml) dilution, ab199975 anti-Aquaporin 2 used at 1 : 4000 (0.143 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1 : 4000 (0.132 ug/ml) dilution.

Panel A : merged staining of anti-NADPH oxidase 4 (green; Opal™520), anti-Aquaporin 2 (magenta; Opal™690) and anti-Angiotensin Converting Enzyme 1 (yellow; Opal™570) on rat kidney.
Panel B : anti-NADPH oxidase 4 staining distal tubules in rat kidney.
Panel C : anti-Aquaporin 2 staining collecting tubules in rat kidney.
Panel D : anti-Angiotensin Converting Enzyme 1 staining proximal tubules in rat kidney.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab319055, ab199975 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-Angiotensin Converting Enzyme 1 antibody [EPR22291-247] (AB254222)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining NADPH oxidase 4 with ab319055 at a 1 : 500 (1.032 ug/ml) dilution, ab233837 anti-Hexokinase 1 used at 1 : 250 (4.36 ug/ml) dilution and ab254222 anti-Angiotensin Converting Enzyme 1 used at a 1 : 4000 (0.132 ug/ml) dilution.

Panel A : merged staining of anti-NADPH oxidase 4 (green; Opal™520), anti-Hexokinase 1 (magenta; Opal™690) and anti-Angiotensin Converting Enzyme 1 (yellow; Opal™570) on mouse kidney.
Panel B : anti-NADPH oxidase 4 staining distal tubules in mouse kidney.
Panel C : anti-Hexokinase 1 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 ab319055, ab233837 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.

• mIHC

Supplier Data

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 ab322047 at a 1 : 2000 (0.261 ug/ml) dilution, 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 ab322047, 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.

• IHC-Fr

Unknown

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) (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).

• IHC-Fr

Unknown

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) (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).

• WB

Lab

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] (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

Predicted band size: 150 kDa

Observed band size: 200 kDa

false

• WB

Supplier Data

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 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).

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 150 kDa

Observed band size: 180 kDa

true

• WB

Supplier Data

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 150 kDa

Observed band size: 180 kDa

false

• WB

Supplier Data

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 150 kDa

Observed band size: 180 kDa

false

• I-ELISA

Unknown

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.