Anti-GPCR GPR97 antibody [RM2090]

• Flow Cyt

Lab

Flow Cytometry - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Flow cytometric analysis of Mouse Blood cells labelling with ab325346 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti mouse CD11b conjugated to Brilliant Violet 421.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Immunohistochemical analysis of paraffin-embedded Mouse bone marrow tissue labeling with ab325346 at 1/2000 (0.249 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) .

Positive staining on mouse bone marrow ( (PMID : 24113187).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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 citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling with ab325346 at 1/2000 (0.249 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) .

Positive staining on mouse spleen ( (PMID : 24113187).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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 citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• Flow Cyt

Lab

Flow Cytometry - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Flow cytometric analysis of Mouse Blood cells labelling with ab325346 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti mouse CD4 conjugated to Alexa Fluor®488.

• Flow Cyt

Lab

Flow Cytometry - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Flow cytometric analysis of Mouse Blood cells labelling with ab325346 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti mouse Gr-1 conjugated to Pacific Blue.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining with ab325346 at a 1 : 5000 (0.099 µg/ml) dilution, ab238132 anti-Ly6g used at 1 : 100 (0.29 µg/ml) dilution and ab237721 anti-CD3 used at a 1 : 2000 (0.26 µg/ml) dilution.

Panel A : anti-GPCR GPR97 (green; Opal™520), anti-Ly6g (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on mouse spleen.
Panel B : anti-GPCR GPR97 staining granulocytes in mouse spleen.
Panel C : anti-Ly6g staining neutrophils in mouse spleen.
Panel D : anti-CD3 staining T lymphocytes in mouse spleen.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325346, ab238132 and ab237721 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

Lab

Immunoprecipitation - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

GPCR GPR97 was immunoprecipitated from 0.35 mg Mouse spleen tissue lysate with ab325346 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325346 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse spleen tissue lysate
Lane 2 : ab325346 IP in Mouse spleen tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325346 in mouse spleen tissue lysate

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

Exposure time : 137 seconds.

All lanes:

Immunoprecipitation - Anti-GPCR GPR97 antibody [RM2090] (ab325346) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 10 µg

Lane 2:

ab325346 at 1/30 IP in Mouse spleen tissue lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325346 in mouse spleen tissue lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa

false

Exposure time: 137s

• WB

Lab

Western blot - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

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

Low expression : brain, thymus, testis, heart (PMID : 24113187).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

Exposure time : Lane 1 : 10 seconds; Lanes 2-6 : 70 seconds.

All lanes:

Western blot - Anti-GPCR GPR97 antibody [RM2090] (ab325346) at 1/1000 dilution

Lane 1:

Mouse bone marrow tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse thymus tissue lysate at 20 µg

Lane 5:

Mouse testis tissue lysate at 20 µg

Lane 6:

Mouse heart 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 1/20000 dilution

Observed band size: 60 kDa,124 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling with ab325346 at 1/2000 (0.099 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) .

Negative control : No staining on mouse cerebrum (PMID : 24113187 ).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling with ab325346 at 1/2000 (0.099 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) .

Negative control : No staining on mouse cardiac muscle (PMID : 24113187 ) .

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

• Flow Cyt

Lab

Flow Cytometry - Anti-GPCR GPR97 antibody [RM2090] (AB325346)

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20 permeabilized 293T cells transfected with a mouse AGRG3 expression vector containing a myc-His-tag® (Middle) 293T cells transfected with an empty expression vector containing a myc-His-tag® (Right) cells labelling with ab325346 at 1/500 dilution (0.1ug) / Right and middle compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells were surface stained with isotype control or our antibody. Then fixed with 4% PFA for 10min followed by intracellularly stained with anti-myc tag conjugated to Alexa Fluor® 647.