Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunohistochemical analysis of paraffin-embedded Human colorectal carcinoma tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colorectal carcinoma.
The section was incubated with ab318284 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 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 membranous and cytoplasmic staining in HUVEC cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : SH-SY5Y.
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 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SH-SY5Y (human neuroblastoma epithelial cell) (Left) / HUVEC (human umbilical vein endothelial cell) (Right) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/500 dilution (0.1ug) / Magenta (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.

Low expression : SH-SY5Y (PMID : 25883214).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on basement membrane of human kidney.
The section was incubated with ab318284 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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon.
The section was incubated with ab318284 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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Heparan Sulfate Proteoglycan 2/Perlecan was immunoprecipitated from 0.35 mg EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate with ab318284 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318284 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate
Lane 2 : ab318284 IP in EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318284 in EA.hy926 whole cell lysate

All lanes:

Immunoprecipitation - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (ab318284) at 1/30 dilution

All lanes:

EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate with NFDM/TBST

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: 76s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (mouse neuroblastoma neuroblast) (Left) / MEF (mouse embryo fibroblast) (Right) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/500 dilution (0.1ug) / Magenta (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.

Low expression : Neuro-2a

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on basement membrane of rat kidney (PMID : 24035513).
The section was incubated with ab318284 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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on basement membrane of mouse kidney (PMID : 23757342).
The section was incubated with ab318284 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab318284 at 1/1000 (0.486 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 cytoplasmic and membranous staining in MEF cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : Neuro-2a.
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 2 ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Low expression : SH-SY5Y (PMID : 25883214).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (ab318284) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 3:

EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate at 20 µg with NFDM/TBST

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: 469 kDa,124 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (AB318284)

Low expression : RAW 264.7, J774A.1(PMID : 35887248), Neuro-2a.

The bands beneath the target band (469 kDa) are likely to be degraded target fragments.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [RM2059] (ab318284) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg with NFDM/TBST

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg with NFDM/TBST

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg with NFDM/TBST

Lane 4:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg with NFDM/TBST

Lane 5:

J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 20 µg with NFDM/TBST

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: 469 kDa,124 kDa

true

Exposure time: 169s