Anti-ISG15 antibody [EPR28318-62]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of paraffin-embedded Mouse cererbrum tissue labeling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : weak staining on mouse cerebrum. The section was incubated with ab315281 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of paraffin-embedded (A) RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours (B) Untreated RAW 264.7 tissue labeling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Raw 264.7 cell pellet treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours; weak staining on (B) Untreated RAW 264.7. The section was incubated with ab315281 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The section was incubated with ab315281 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-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of paraffin-embedded Mouse glioblastoma tissue labeling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse glioblastoma. The section was incubated with ab315281 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-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of paraffin-embedded Mouse breast cancer tissue labeling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse breast cancer. The section was incubated with ab315281 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-Fr

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Immunohistochemistry (Frozen sections) - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling ISG15 with ab315281 at 1/500 (1.048 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on Mousee spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab315281 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling ISG15 with ab315281 at 1/2000 (0.262 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing increased cytoplasmic staining in RAW 264.7 cells treated with Lipopolysaccharide (LPS, 1 μg/mL) for 24 hours, then add Brefeldin A (BFA, 1 μg/mL) for 3 hours. 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 (2.5 ug/ml) dilution (Red). 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 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ISG15 antibody [EPR28318-62] (AB315281)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1 µg/mL LPS for 24h and 1ug/ml BFA for 3 h (Red) / untreated RAW 264.7 (Green) cells labelling ISG15 with ab315281 at 1/5000 dilution (0.01 ug, 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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-ISG15 antibody [EPR28318-62] (AB315281)

ISG15 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours whole cell lysate with ab315281 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315281 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours whole cell lysate

Lane 2 : ab315281 IP in RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab315281 in RAW 264.7 treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours whole cell lysate

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16815975).

All lanes:

Immunoprecipitation - Anti-ISG15 antibody [EPR28318-62] (ab315281) at 1/30 dilution

All lanes:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 1 ug/ml Lipopolysaccharides (LPS) for 21 hours, 1 ug/ml Brefeldin A was then added for additional 3 hours 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: 10s

• WB

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Western blot - Anti-ISG15 antibody [EPR28318-62] (AB315281)

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

The expression of ISG15 is upregulated in response to Lipopolysaccharides treatment (PMID : 11854279).

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

All lanes:

Western blot - Anti-ISG15 antibody [EPR28318-62] (ab315281) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 1 μg/ml Lipopolysaccharides (LPS) for 21 hours, 1 μg/ml Brefeldin A was then added for additional 3 hours 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: 17 kDa,36 kDa

false

Exposure time: 10s