Anti-MPTX+MPTX2 antibody [EPR20920-19]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) transfected with a flag-tagged MPTX expression construct cells labeling MPTX1 with ab238123 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing positive staining in HEK-293T cells transfected with a flag-tagged MPTX expression construct.

Mouse monoclonal anti-DDDDK antibody followed by ab150120 AlexaFluor^®594 Goat anti-Mouse secondary was used to counterstain at 1/500 dilution (Red). The nuclear counterstain was DAPI (Blue).

-ve control 1 : ab238123 at a 1/50 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : Mouse monoclonal anti-DDDDK antibody at a 1/500 dilution followed by ab150077 at a 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney epithelial cell) transfected with a flag-tagged MPTX expression construct cells labeling MPTX1 with ab238123 at 1/500 (Right panel) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left panel). A Goat anti rabbit IgG (Alexa Fluor^® 647, ab150079) at 1/2000 was used as the secondary antibody.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen mouse small intestine tissue labeling MPTX1 with ab238123 at 1/500 (1.10 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). 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.

Positive staining on Paneth cells in mouse small intestine is observed (PMID : 29144463).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining Eph receptor B2 with ab252935 at 1/500 dilution, ab314897 anti-CaMKII beta used at 1/5000 dilution and ab238123 anti-MPTX used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

Panel A : merged staining of anti-Eph receptor B2 (green; Opal™520), anti-CaMKII beta (magenta; Opal™690) and anti-MPTX (gray; Opal™570) on mouse small intestine.
Panel B : anti-Eph receptor B2 staining membrane of stem cells in mouse small intestine.
Panel C : anti-CaMKII beta staining endocrine cells in mouse small intestine.
Panel D : anti-MPTX staining membrane of Paneth cells in mouse small intestine.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab259935, ab314897 and ab238123 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue labeling MPTX1 with ab238123 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on Paneth cells of mouse small intestine is observed. The section was incubated with ab238123 for 5 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control : Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with citrate buffer (pH 6.0, epitope retrieval solution 1) for 10mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining CaMKII beta with ab314897 at a 1/5000 dilution, ab315150 anti-Serotonin used at 1/5000 dilution and ab238123 anti-MPTX used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

Panel A : merged staining of anti-CaMKII beta (green; Opal^™690), anti-Serotonin (magenta; Opal^™520) and anti-MPTX (grey; Opal^™570) on mouse small intestine.
Panel B : anti-CaMKII beta staining myenteric nerve plexus and endocrine cells in mouse small intestine.
Panel C : anti-Serotonin staining enterochromaffin cells in mouse small intestine.
Panel D : anti-MPTX staining membrane of Paneth cells in mouse small intestine.
Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining : in the order of ab314897, ab315150 and ab238123 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.

• WB

Lab

Western blot - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

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

MPTX2 is reported to express in Paneth cells in the small intestine (PMID : 29144463).

The lysates were kindly provided by an anonymous collaborator.

ab238123 was shown to specifically react with MPTX in wild-type mouse intestine tissue as signal was lost in MPTX knockout tissue. Wild-type and MPTX knockout samples were subjected to SDS-PAGE. ab238123 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™ instrument using the ECL technique. Exposure time : 15 seconds.

All lanes:

Western blot - Anti-MPTX+MPTX2 antibody [EPR20920-19] (ab238123) at 1/1000 dilution

Lane 1:

Wild type mouse large intestine tissue lysate at 10 µg

Lane 2:

MPTX knockout mouse large intestine tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

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• WB

Lab

Western blot - Anti-MPTX+MPTX2 antibody [EPR20920-19] (AB238123)

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

MPTX2 protein carries a lif tag (MW around 40 kDa).

Exposure time : 3 seconds.

All lanes:

Western blot - Anti-MPTX+MPTX2 antibody [EPR20920-19] (ab238123) at 1/1000 dilution

Lane 1:

His-tagged mouse MPTX recombinant protein, 10ng

Lane 2:

His-lif tagged mouse MPTX2 recombinant protein, 10ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

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