Anti-PVRIG/CD112R antibody [EPR29052-91]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling PVRIG/CD112R with ab322348 at 1/100 (5.16 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human skeletal muscle. The section was incubated with ab322348 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

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human colon staining of PVRIG/CD112R with ab322348 at a 1/100 (5.16 µg/ml) dilution, CD19 with ab320735 at 1/2000 (0.255 µg/ml) dilution and CD8A with ab245118 at 1/1000 ( 0.375 µg/ml) dilution.

Panel A : merged staining of anti-PVRIG/CD112R (green; Opal™520), anti-CD8A (magenta; Opal™570) and anti-CD19 (yellow; Opal™690) on Human colon.
Panel B : anti-PVRIG/CD112R staining memory and effector CD8+ T lymphocytes in Human colon.
Panel C : anti-CD8A staining CD8+ T lymphocytes in Human colon.
Panel D : anti-CD19 staining B lymphocytes in Human colon.
Nuclear DNA was labelled with DAPI (shown in blue). ).

The section was incubated in three rounds of staining : in the order of ab322348, ab245118 and ab320735 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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human spleen staining of PVRIG/CD112R with ab322348 at a 1/100 (5.16 µg/ml) dilution, CD19 with ab320735 at 1/2000 (0.255 µg/ml) dilution and CD8A with ab245118 at 1/1000 ( 0.375 µg/ml) dilution.

Panel A : merged staining of anti-PVRIG/CD112R (green; Opal™520), anti-CD8A (magenta; Opal™570) and anti-CD19 (yellow; Opal™690) on human spleen.
Panel B : anti-PVRIG/CD112R staining memory and effector CD8+ T lymphocytes in human spleen.
Panel C : anti-CD8A staining CD8+ T lymphocytes in human spleen.
Panel D : anti-CD19 staining B lymphocytes in human spleen.
Nuclear DNA was labelled with DAPI (shown in blue). ).

The section was incubated in three rounds of staining : in the order of ab322348, ab245118 and ab320735 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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human tonsil staining of PVRIG/CD112R with ab322348 at a 1/100 (5.16 µg/ml) dilution, CD19 with ab320735 at 1/2000 (0.255 µg/ml) dilution and CD8A with ab245118 at 1/1000 ( 0.375 µg/ml) dilution.

Panel A : merged staining of anti-PVRIG/CD112R (green; Opal™520), anti-CD8A (magenta; Opal™570) and anti-CD19 (yellow; Opal™690) on human tonsil.
Panel B : anti-PVRIG/CD112R staining memory and effector CD8+ T lymphocytes in human tonsil.
Panel C : anti-CD8A staining CD8+ T lymphocytes in human tonsil.
Panel D : anti-CD19 staining B lymphocytes in human tonsil.
Nuclear DNA was labeled with DAPI (shown in blue). ).

The section was incubated in three rounds of staining : in the order of ab322348, ab245118 and ab320735 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling PVRIG/CD112R with ab322348 at 1/100 (5.16 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The section was incubated with ab322348 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-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling PVRIG/CD112R with ab322348 at 1/100 (5.16 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human spleen. The section was incubated with ab322348 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-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling PVRIG/CD112R with ab322348 at 1/100 (5.16 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab322348 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-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type Jurkat (Human T lymphocyte) cell pellet and (B) Human PVRIG knockout Jurkat cell pellet labeling PVRIG/CD112R with ab322348 at 1/100 (5.16 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) wild-type Jurkat cell pellet, no staining on (B) human PVRIG knockout Jurkat cell pellet. The section was incubated with ab322348 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

• IP

Supplier Data

Immunoprecipitation - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

PVRIG/CD112R was immunoprecipitated from 0.35 mg Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate with ab322348 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322348 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate
Lane 2 : ab322348 IP in Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322348 in Daudi whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-PVRIG/CD112R antibody [EPR29052-91] (ab322348) at 1/30 dilution

All lanes:

Daudi (human Burkitt's lymphoma lymphoblast) whole cell 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

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

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

Negative control : HeLa

Low expression : MOLT-4, Raji (PMID : 26755705)

This blot of lane 6 was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

The identity of the bands below 30- 40 kDa in lane 2 and lane 3 are unknown.

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

Exposure time : Lanes 1-5 : 92 seconds; Lane 6 : 180 seconds.

All lanes:

Western blot - Anti-PVRIG/CD112R antibody [EPR29052-91] (ab322348) at 1/1000 dilution

Lane 1:

Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 2:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 5:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 6:

Human lymph node 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 1/100000 dilution

Observed band size: 30-40 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-PVRIG/CD112R antibody [EPR29052-91] (AB322348)

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

Performed under reducing conditions.

In Western blot, ab322348 was shown to bind specifically to PVRIG. Target of interest was observed at 30-40 kDa in wild-type Jurkat cell lysates (lane 1) with no signal observed at this size in PVRIG knockout cell line (lane 2) (lane 2, knockout cell line ab274900).

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

All lanes:

Western blot - Anti-PVRIG/CD112R antibody [EPR29052-91] (ab322348) at 1/1000 dilution

Lane 1:

Wild-type Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 40 µg

Lane 2:

PVRIG knockout Jurkat whole cell lysate at 40 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: 30-40 kDa,36 kDa

false

Exposure time: 103s