Anti-Thy1.2 antibody [EPR31143-526]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Thy1.2 antibody [EPR31143-526] (AB325921)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining with ab325921 at a 1 : 2000 (0.25 µg/ml) dilution, ab300421 anti-F4/80 used at 1 : 5000 (0.106 µg/ml) dilution and ab64088 anti-CD20 used at a 1 : 100 (0.11 µg/ml) dilution.

Panel A : anti-Thy1.2 (green; Opal™520), anti-F4/80 (magenta; Opal™690), anti-CD20 (gray; Opal™570) on mouse spleen.
Panel B : anti-Thy1.2 staining T lymphocytes in mouse spleen.
Panel C : anti-F4/80 staining macrophages in mouse spleen.
Panel D : anti-CD20 staining B 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 ab325921, ab300421 and ab64088 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.2 antibody [EPR31143-526] (AB325921)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling with ab325921 at 1/2000 (0.25 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in 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-Thy1.2 antibody [EPR31143-526] (AB325921)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling with ab325921 at 1/2000 (0.25 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in 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

• WB

Lab

Western blot - Anti-Thy1.2 antibody [EPR31143-526] (AB325921)

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

Negative control : skeletal muscle(PMID : 74016)

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

All lanes:

Western blot - Anti-Thy1.2 antibody [EPR31143-526] (ab325921) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse skeletal muscle 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: 25 kDa,36 kDa

false

Exposure time: 37s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thy1.2 antibody [EPR31143-526] (AB325921)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling with ab325921 at 1/2000 (0.25 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining in mouse skeletal 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.

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

• WB

Lab

Western blot - Anti-Thy1.2 antibody [EPR31143-526] (AB325921)

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

This antibody does not cross-react with overexpressed mouse Thy1.1 by western blot.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(37,55kDa).

All lanes:

Western blot - Anti-Thy1.2 antibody [EPR31143-526] (ab325921) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with mouse Thy1.1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T cells transfected with mouse Thy1.2 expression vector containing a myc-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 1/100000 dilution

Observed band size: 25 kDa,36 kDa,37 kDa,55 kDa

false

Exposure time: 48s