Anti-UMOD antibody [EPR22887-217]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-UMOD antibody [EPR22887-217] (AB255825)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat kidney tissue staining UMOD with ab255825 at 1/1000 (0.5 μg/ml), ab313787 anti-Nestin, and ab326049 anti-Renin-1+ Renin-2.

Panel A : anti-UMOD (green; Opal™520), anti-Nestin (magenta; Opal™690), anti-Renin-1+Renin-2 (gray; Opal™570) on rat kidney.

Panel B : anti-UMOD staining the thick ascending limb of the loop of Henle (TALH) in rat kidney.

Panel C : anti-Nestin staining glomerulus and endothelium in rat kidney.

Panel D : anti-Renin-1+Renin-2 staining juxtaglomerular cells 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 ab255825, ab313787 and ab326049 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-UMOD antibody [EPR22887-217] (AB255825)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining UMOD with ab255825 at 1/1000 (0.5 μg/ml), ab229037 anti-NPHS2, and ab326049 anti-Renin-1+ Renin-2.

Panel A : anti-UMOD (green; Opal™520), anti-NPHS2 (magenta; Opal™690), anti-Renin-1+Renin-2 (gray; Opal™570) on mouse kidney.

Panel B : anti-UMOD staining the thick ascending limb of the loop of Henle (TALH) in mouse kidney.

Panel C : anti-NPHS2 staining glomerular podocyte in mouse kidney.

Panel D : anti-Renin-1+Renin-2 staining juxtaglomerular cells 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 ab255825, ab229037 and ab326049 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-UMOD antibody [EPR22887-217] (AB255825)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat kidney tissue staining UMOD with ab255825 at 1/1000 (0.5 μg/ml), ab229037 anti-NPHS2, and ab326049 anti-Renin-1+ Renin-2.

Panel A : anti-UMOD (green; Opal™520), anti-NPHS2 (magenta; Opal™690), anti-Renin-1+Renin-2 (gray; Opal™570) on rat kidney.

Panel B : anti-UMOD staining the thick ascending limb of the loop of Henle (TALH) in rat kidney.

Panel C : anti-NPHS2 staining glomerular podocyte in rat kidney.

Panel D : anti-Renin-1+Renin-2 staining juxtaglomerular cells 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 ab255825, ab229037 and ab326049 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-UMOD antibody [EPR22887-217] (AB255825)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse kidney tissue staining UMOD with ab255825 at 1/1000 (0.5 μg/ml), ab313787 anti-Nestin, and ab326049 anti-Renin-1+ Renin-2.

Panel A : anti-UMOD (green; Opal™520), anti-Nestin (magenta; Opal™690), anti-Renin-1+Renin-2 (gray; Opal™570) on mouse kidney.

Panel B : anti-UMOD staining the thick ascending limb of the loop of Henle (TALH) in mouse kidney.

Panel C : anti-Nestin staining glomerulus and endothelium in mouse kidney.

Panel D : anti-Renin-1+Renin-2 staining juxtaglomerular cells 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 ab255825, ab313787 and ab326049 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UMOD antibody [EPR22887-217] (AB255825)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Umod with ab255825 at 1/ 2000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) Kidney tissue from wild-type C57BL/6J mice, no staining on (B) Kidney tissue from Umod knockout mice. The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Umod-KO homozygous mice (Strain ID : T014822).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-UMOD antibody [EPR22887-217] (AB255825)

Immunohistochemical analysis of 4% PFA fixed, 0.2% Triton X-100 permeabilized frozen Mouse kidney tissue labeling UMOD with ab255825 at 1/100 (5.00 μg/ml) dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Positive staining on the thick ascending limb of the loop of Henle (TALH) in mouse kidney (PMID : 28781372) is observed.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UMOD antibody [EPR22887-217] (AB255825)

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling UMOD with ab255825 at 1/100 dilution (5.0 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on the thick ascending limb of the loop of Henle (TALH) in rat kidney (PMID : 14569098) is observed. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control/ Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UMOD antibody [EPR22887-217] (AB255825)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling UMOD with ab255825 at 1/100 dilution (5.0 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on the thick ascending limb of the loop of Henle (TALH) in mouse kidney (PMID : 14569098) is observed. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-UMOD antibody [EPR22887-217] (AB255825)

Immunohistochemical analysis of 4% PFA fixed, 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labeling UMOD with ab255825 at 1/100 (5.00 μg/ml) dilution followed by ab150077 AlexaFluora 488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Positive staining on the thick ascending limb of the loop of Henle (TALH) in rat kidney (PMID : 28781372) is observed.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/ml) dilution.

• IP

Unknown

Immunoprecipitation - Anti-UMOD antibody [EPR22887-217] (AB255825)

UMOD was immunoprecipitated from 0.35 mg Mouse urine with ab255825 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab255825 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse urine 10μg

Lane 2 : ab255825 IP in Mouse urine

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab255825 in Mouse urine.

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

Exposure time : 50 seconds.

All lanes:

Immunoprecipitation - Anti-UMOD antibody [EPR22887-217] (ab255825)

Predicted band size: 70 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-UMOD antibody [EPR22887-217] (AB255825)

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

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

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Umod-KO homozygous mice (Strain ID : T014822).

All lanes:

Western blot - Anti-UMOD antibody [EPR22887-217] (ab255825) at 1/1000 dilution

Lane 1:

Wild-type mouse kidney tissue lysate (male) at 5 µg

Lane 2:

Wild-type mouse kidney tissue lysate (female) at 5 µg

Lane 3:

Umod knockout mouse kidney tissue lysate (male case1) at 5 µg

Lane 4:

Umod knockout mouse kidney tissue lysate (male case2) at 5 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 1/20000 dilution

Observed band size: 100-120 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-UMOD antibody [EPR22887-217] (AB255825)

UMOD is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID : 24097801).

Exposure time : Lane 1 : 10 seconds; Lanes 2-3 : 3 minutes.

Blocking and Diluting Buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-UMOD antibody [EPR22887-217] (ab255825) at 1/1000 dilution

Lane 1:

Rat urine at 20 µg

Lane 2:

Mouse kidney lysate at 20 µg

Lane 3:

Mouse urine 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: 55 kDa,70 kDa

Observed band size: 100-120 kDa

false