Anti-Sp8 antibody [EPR29574-561]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocytes labelling Sp8 with ab324637 at 1/50 (9.78 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Negative control : confocal image showing no staining in mouse splenocytes (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-mouse CD3 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Magenta).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling Sp8 with ab324637 at 1/50 (9.78 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Negative control : confocal image showing no staining on mouse spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab324637 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of paraffin-embedded Mouse brain tissue labeling Sp8 with ab324637 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse brain with intense staining in the olfactory bulb. 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 only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed 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-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Sp8 with ab324637 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on mouse lung (PMID : 15533246). 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 only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Sp8 with ab324637 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on mouse spleen (PMID : 15533246). 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 only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Sp8 with ab324637 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on mouse liver (PMID : 15533246). 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 only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NCI-H69 (human lung small cell carcinoma cell line); U-937 (human histiocytic lymphoma monocyte) cells labelling Sp8 with ab324637 at 1/50 (9.78 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing nuclear staining in NCI-H69 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Negative control : U-937.

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab324637 at a 1/50 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab7291 at a 1/1000 dilution followed by ab150081 at a 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse primary olfactory bulb neurons labelling Sp8 with ab324637 at 1/50 (9.78 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing nuclear staining in mouse primary olfactory bulb neurons (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 (4 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab324637 at a 1/50 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab11267 at a 1/500 dilution followed by ab150081 at a 1/1000 dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Sp8 antibody [EPR29574-561] (AB324637)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse olfactory bulb (fresh frozen) tissue labeling Sp8 with ab324637 at 1/50 (9.78 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Panel A : merged staining of anti-SP8 (ab324637, green), anti-NeuN (ab190565, white) and anti-GFAP (ab201732, magenta) on mouse olfactory bulb.
Panel B : anti-SP8 stained on mouse olfactory bulb.
Panel C : anti-NeuN stained in neurons of mouse olfactory bulb.
Panel D : anti-GFAP stained in astrocytes of mouse olfactory bulb.
The section was incubated in two rounds of staining : in the order of ab324637 and ab190565, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution.

• WB

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Western blot - Anti-Sp8 antibody [EPR29574-561] (AB324637)

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

Low expression : U-937.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Sp8 antibody [EPR29574-561] (ab324637) at 1/1000 dilution

Lane 1:

NCI-H69 (human lung small cell carcinoma cell line) whole cell lysate at 50 µg

Lane 2:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 50 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: 50 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Sp8 antibody [EPR29574-561] (AB324637)

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

Low expression : brain, liver.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Sp8 antibody [EPR29574-561] (ab324637) at 1/1000 dilution

Lane 1:

Mouse olfactory bulb tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 50 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Sp8 antibody [EPR29574-561] (AB324637)

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

This antibody does not cross-react with mouse SP9.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Sp8 antibody [EPR29574-561] (ab324637) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse SP8 expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse SP9 expression vector containing a His-tag, 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 1/20000 dilution

Observed band size: 50 kDa,20-50 kDa,36 kDa

false

Exposure time: 6s