Anti-DGKZ/DGK-zeta antibody [EPR22040-80]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human cerebellum staining of Calbindin with ab108404 at a 1/1000 dilution, DGKZ/DGK-zeta with ab239081 at 1/5000 dilution, and GABA A Receptor alpha 6 with ab300069 at a 1/500 dilution, followed by a ready to use secondary antibody Opal Polymer HRP Ms + Rb.

Panel A : merged staining of anti-GABA A Receptor alpha 6 (gray; Opal™690), anti-DGKZ/DGK-zeta (green; Opal™520) and anti-Calbindin (magenta; Opal™570) on human cerebellum.
Panel B : anti-Calbindin stained on all layers of cerebellum.
Panel C : anti-DGKZ/DGK-zeta stained on Purkinje cells.
Panel D : anti-GABA A Receptor alpha 6 stained on the granular layer.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab300069, ab239081, and ab108404 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.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling DGKZ/DGK-zeta with ab239081 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on Purkinje cells of human cerebellum (PMID : 14511325; PMID : 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunofluorescent analysis of 100% methanol-fixed Jurkat (human T cell leukemia cell line from peripheral blood) cells labeling DGKZ/DGK-zeta with ab239081 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining in Jurkat cell line.

Methanol is recommended for fixation as weak signal was observed using 4% PFA.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Jurkat (human T cell leukemia cell line from peripheral blood) cell line labeling DGKZ/DGK-zeta with ab239081 at 1/600 (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunofluorescent analysis of 100% methanol-fixed HuT-78 (human Sezary syndrome cutaneous T lymphocyte cell line) cells labeling DGKZ/DGK-zeta with ab239081 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining in HuT-78 cell line.

Methanol is recommended for fixation as weak signal was observed using 4% PFA.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling DGKZ/DGK-zeta with ab239081 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on subset of immune cells in human tonsil (PMID : 24573202) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labeling DGKZ/DGK-zeta with ab239081 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on Purkinje cells of rat cerebellum (PMID : 14511325; PMID : 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labeling DGKZ/DGK-zeta with ab239081 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on Purkinje cells of mouse cerebellum (PMID : 14511325; PMID : 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (AB239081)

Exposure time : Lane 1 : 3 seconds; Lanes 2 & 7 : 10 seconds; Lanes 3-6 : 37 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

The lysates were no-boiled to avoid the protein aggregation.

Multiple bands observed are likely due to isoforms and phosphorylation. The molecular mass of human DGKZ / DGK-zeta is also predicated to be larger than that of rodents. The molecular profile/weight observed is consistent with what has been described in the literature (PMID : 12070163, PMID : 14511325, PMID : 9657393).

All lanes:

Western blot - Anti-DGKZ/DGK-zeta antibody [EPR22040-80] (ab239081) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

HuT-78 (human Sezary syndrome cutaneous T lymphocyte cell line) whole cell lysate at 20 µg

Lane 3:

Mouse brain lysate at 20 µg

Lane 4:

Mouse cerebellum lysate at 20 µg

Lane 5:

Mouse thymus lysate at 20 µg

Lane 6:

Rat brain lysate at 20 µg

Lane 7:

Rat cerebellum 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 1/50000 dilution

Predicted band size: 124 kDa

Observed band size: 90-130 kDa

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