Anti-GM-CSF antibody [EPR28743-78]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling GM-CSF with ab316862 at 1/200 (2.54 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human lung (PMID : 32719685).
The section was incubated with ab316862 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HuT-78 (human Sezary syndrome cutaneous T lymphocyte) cells labelling GM-CSF with ab316862 at 1/50 (10.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in HuT-78 cells treated with Phorbol-12-myristate-13-acetate (80 nM) and Ionomycin (3 uM) for 5 hours, then add Brefeldin A (300 ng/ml) for 4 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HuT-78(human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 3uM onomycin for 5h and add 300ng/ml BFA for 4h (Red)/Untreated HuT-78 (Dotted red) cells labelling GM-CSF with ab316862 at 1/5000 dilution (0.01ug)/Red and dotted red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 488, ab150077) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling GM-CSF with ab316862 at 1/200 (2.54 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human colon carcinoma (PMID : 31908491).
The section was incubated with ab316862 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-GM-CSF antibody [EPR28743-78] (AB316862)

Immunohistochemical analysis of paraffin-embedded (A) HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with TPA (80nM/5 hours) and ionomycin (30µM/5 hours) add BFA (300ngml/4 hours). (A) HuT-78 untreated with TPA (80nM/5 hours) and ionomycin (30µM/5 hours) add BFA (300ngml/4 hours). tissue labeling GM-CSF with ab316862 at 1/2000 (0.254 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HuT-78 treated with TPA (80nM/5 hours) and ionomycin (30µM/5 hours) add BFA (300ngml/4 hours). No staining on (B) HuT-78 untreated with TPA (80nM/5 hours) and ionomycin (30µM/5 hours) add BFA (300ngml/4 hours).
The section was incubated with ab316862 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

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Immunoprecipitation - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

GM-CSF was immunoprecipitated from 0.35 mg HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 30uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, whole cell lysate with ab316862 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316862 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 30uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, whole cell lysate
Lane 2 : ab316862 IP in HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 30uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316862 in HuT-78 treated with 80nM TPA and 30uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h whole cell lysate

All lanes:

Immunoprecipitation - Anti-GM-CSF antibody [EPR28743-78] (ab316862) at 1/30 dilution

All lanes:

HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 30uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, 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

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Western blot - Anti-GM-CSF antibody [EPR28743-78] (AB316862)

The identity of the higher MW band at approximately 50 kDa is unknown.

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

All lanes:

Western blot - Anti-GM-CSF antibody [EPR28743-78] (ab316862) at 1/1000 dilution

Lane 1:

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

Lane 2:

HuT-78 treated with 80nM TPA and 30uM Ionomycin for 5h, /ml BFA was then added for additional 4h, 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: 20 kDa,36 kDa

false

Exposure time: 37s