Anti-Thy1.1 antibody [EPR31142-29]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunohistochemical analysis of paraffin-embedded (A) FVB/NCrl strain mouse thymus and (B) ICR (CD-1) strain mouse thymus tissue labeling with ab326876 at 1/100 (4.79 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) FVB/NCrl strain mouse thymus, no staining on (B) ICR (CD-1) strain mouse thymus.

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunohistochemical analysis of paraffin-embedded FVB/NCrl strain mouse cardiac muscle tissue labeling with ab326876 at 1/100 (4.79 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on FVB/NCrl strain mouse cardiac muscle.

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.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized FVB/NCrl strain mouse splenocytes and C57BL/6J strain mouse splenocytes cells labelling with ab326876 at 1/50 (9.58 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in FVB/NCrl strain mouse splenocytes(shown in green), which is co-stained with the CD3 marker. 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 CD3 at 1/100 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• Flow Cyt

Lab

Flow Cytometry - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Flow cytometric analysis of FVB/NCrl mouse spleen cell labelling with ab326876 at 1/5000 dilution (0.01ug) / 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® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with CD3 conjugated to Alexa Fluor®647.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunohistochemical analysis of paraffin-embedded (A) FVB/NCrl strain mouse cerebrum and (B) ICR (CD-1) strain mouse cerebrum tissue labeling with ab326876 at 1/100 (4.79 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) FVB/NCrl strain mouse cerebrum, no staining on (B) ICR (CD-1) strain mouse cerebrum.

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunohistochemical analysis of paraffin-embedded
(A) HEK-293T (human embryonic kidney epithelial cell) transfected with a Thy1(Q108R) (corresponding to Thy1.1 protein) expression vector containing a his tag
(B) HEK-293T (human embryonic kidney epithelial cell) transfected with a Thy1 expression vector (corresponding to Thy1.1 protein) containing a his tag
(C) HEK-293T cells transfected with empty vector containing a myc-His-tag
labeling with ab326876 at 1/100 (4.79μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a Thy1(Q108R) (corresponding to Thy1.1 protein) expression vector containing a his tag, no staining on (B) HEK-293T (human embryonic kidney epithelial cell) transfected with a Thy1 expression vector (corresponding to Thy1.1 protein) containing a his tag and (C) HEK-293T cells transfected with empty vector containing a myc-His-tag.

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Immunohistochemical analysis of paraffin-embedded (A) FVB/NCrl strain mouse spleen and (B) ICR (CD-1) strain mouse spleen tissue labeling with ab326876 at 1/100 (4.79 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) FVB/NCrl strain mouse spleen, no staining on (B) ICR (CD-1) strain mouse spleen.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Flow cytometric analysis of FVB/NCrl mouse spleen cell labelling with ab326876 at 1/5000 dilution (0.01ug) / 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® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with CD19 conjugated to PE/CY7.

• IP

Lab

Immunoprecipitation - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Thy1.1 was immunoprecipitated from 0.35 mg FVB/NCrl strain mouse thymus tissue lysate with ab326876 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326876 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : FVB/NCrl strain mouse thymus tissue lysate
Lane 2 : ab326876 IP in FVB/NCrl strain mouse thymus tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326876 in FVB/NCrl strain mouse thymus tissue lysate

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

Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-Thy1.1 antibody [EPR31142-29] (ab326876) at 1/1000 dilution

Lane 1:

FVB/NCrl strain mouse thymus tissue lysate at 10 µg

Lane 2:

ab326876 at 1/30 IP in FVB/NCrl strain mouse thymus tissue lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab326876 in FVB/NCrl strain mouse thymus tissue lysate

Secondary

All lanes:

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

Observed band size: 25 kDa

false

Exposure time: 15s

• I-ELISA

Lab

Indirect ELISA - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Indirect ELISA analysis of ab326876 at 1000-0 ng/ml. The Secondary antibody used was Peroxidase AffiniPure Goat Anti-Rabbit IgG, Fc fragment specific at 1 : 20000 dilution dilution.

Antigen : Mouse Thy1.1 protein;Mouse Thy1.2 protein.

Antigen concentration : 1000 ng/ml

This antibody does not cross-react with Mouse Thy1.2 protein by ELISA.

• WB

Lab

Western blot - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Thy1.1 antibody [EPR31142-29] (ab326876) at 1/1000 dilution

Lane 1:

FVB/NCrl strain mouse spleen tissue lysate at 48 µg

Lane 2:

FVB/NCrl strain mouse thymus tissue lysate at 48 µg

Lane 3:

FVB/NCrl strain mouse heart tissue lysate at 48 µg

Lane 4:

FVB/NCrl strain mouse skeletal muscle tissue lysate at 48 µg

Lane 5:

C57BL/6J strain mouse spleen tissue lysate at 48 µg

Lane 6:

C57BL/6J strain mouse thymus tissue lysate at 48 µg

Lane 7:

ICR (CD-1) strain mouse spleen tissue lysate at 48 µg

Lane 8:

ICR (CD-1) strain mouse thymus tissue lysate at 48 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 1/20000 dilution

Observed band size: 25 kDa,36 kDa

false

Exposure time: 59s

• Flow Cyt

Lab

Flow Cytometry - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Flow cytometric analysis of C57BL/6J mouse spleen cell labelling with ab326876 at 1/5000 dilution (0.01ug) / 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® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with CD3 conjugated to Alexa Fluor®647.

• Flow Cyt

Lab

Flow Cytometry - Anti-Thy1.1 antibody [EPR31142-29] (AB326876)

Flow cytometric analysis of C57BL/6J mouse spleen cell labelling with ab326876 at 1/5000 dilution (0.01ug) / 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® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were co-stained with CD19 conjugated to PE/CY7.