Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse breast tissue staining Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at a 1/100 dilution, ab324191 anti-E Cadherin used at 1/500 dilution and ab316115 anti-Perilipin-1 used at a 1/2000 dilution.

Panel A : merged staining of anti-Perlecan (green; Opal™520), anti-E Cadherin (magenta; Opal™690) and anti-Perilipin-1 (gray; Opal™570) on mouse breast.

Panel B : anti-Perlecan staining endothelium and basement membrane in mouse breast.

Panel C : anti-E Cadherin staining epithelium in mouse breast.

Panel D : anti-Perilipin-1 staining adipocytes in mouse breast.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab325029, ab324191 and ab316115 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

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.

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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Immunohistochemical analysis of paraffin-embedded (A) NIH/3T3 (mouse embryonic fibroblast ) cell pellets; (B) RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cell pellets. tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/500 (0.988 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) NIH/3T3 (mouse embryonic fibroblast ) cell pellets, and no staining on (B) RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cell pellets.The section was incubated with ab315029 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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse kidney (fresh) tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/500 (0.988 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 mouse kidney. The nuclear counterstain was DAPI (Blue). The section was incubated with ab315029 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) preadsorbedat 1/1000 (2 ug/mL) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/100 (4.94 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on basement membrane in mouse kidney.The section was incubated with ab315029 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 [EPR28346-84] (AB315029)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/100 (4.94 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on basement membrane in mouse liver.The section was incubated with ab315029 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

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Immunocytochemistry/ Immunofluorescence - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/2000 (0.247 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 staining in MEF cell line.

Negative control : 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 (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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MEF (mouse embryo fibroblast) cells labelling Heparan Sulfate Proteoglycan 2/Perlecan with ab315029 at 1/500 dilution (0.1 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

Supplier Data

Immunoprecipitation - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

Heparan Sulfate Proteoglycan 2/Perlecan was immunoprecipitated from 0.35 mg MEF (mouse embryo fibroblast) whole cell fresh lysate with ab315029 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315029 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : MEF (mouse embryo fibroblast) whole cell fresh lysate

Lane 2 : ab315029 IP in MEF (mouse embryo fibroblast) whole cell fresh lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab315029 in MEF whole cell fresh lysate

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

Lysates were made fresh and used immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (ab315029) at 1/30 dilution

All lanes:

MEF (mouse embryo fibroblast) whole cell fresh 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-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

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

Negative control : Neuro-2a.

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

All lanes:

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (ab315029) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell fresh lysate at 50 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell fresh lysate at 50 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 1/20000 dilution

Observed band size: 469 kDa,124 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (AB315029)

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

Negative control : RAW 264.7(PMID : 35887248).

In blot 2, the lysate was stored at -80℃ prior to Western Blotting. In blot 1, lysate was freshly made and used for Western Blotting immediately to minimize protein degradation.

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

All lanes:

Western blot - Anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody [EPR28346-84] (ab315029) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 50 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 50 µg

Lane 3:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 50 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 1/20000 dilution

Observed band size: 469 kDa,124 kDa

false

Exposure time: 48s