Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling SQSTM1 with ab314504 at 1/5000 (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer.The section was incubated with ab314504 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized SQSTM1 KO HAP1(SQSTM1 knockout human chronic myelogenous leukemia cell line) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 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 cytoplasmic staining in HAP1 cell line, and negative staining in SQSTM1 KO HAP1 cell line. 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 2ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HCT116 (human colon epithelial) cell pellet (B) SQSTM1 knockout HCT116 (ab266871) cell pellet tissue labeling SQSTM1 with ab314504 at 1/20000 (0.025 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) Wild-type HCT116 cell pellet, no staining on (B) SQSTM1 knockout HCT116 (ab266871) cell pellet. The section was incubated with ab314504 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SQSTM1 KO HAP1 (human SQSTM1 knockout chronic myelogenous leukemia near-haploid cell) / parental HAP1 cells labelling SQSTM1 with ab314504 at 1/5000 dilution (0.01 ug)/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). A 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-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

SQSTM1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab314504 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314504 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate Lane 2 : ab314504 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314504 in HepG2 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (ab314504) at 1/30 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell) 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: 62s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast cell) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 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 cytoplasmic staining in MEF cell line. 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 2ug/ml dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 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 cytoplasmic staining in PC-12 cell line. 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 2ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling SQSTM1 with ab314504 at 1/1000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum.The section was incubated with ab314504 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh) tissue labeling SQSTM1 with ab314504 at 1/50 (10.1 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 cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab314504 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-Fr

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Immunohistochemistry (Frozen sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (fresh) tissue labeling SQSTM1 with ab314504 at 1/50 (10.1 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 rat cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab314504 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling SQSTM1 with ab314504 at 1/1000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat cerebrum.The section was incubated with ab314504 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling SQSTM1 with ab314504 at 1/5000 dilution (0.01 ug)/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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MEF (mouse embryo fibroblast) cells labelling SQSTM1 with ab314504 at 1/5000 dilution (0.01 ug)/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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-SQSTM1 / p62 antibody (ab314504) staining at 1/1000 dilution, shown in green; Mouse Anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20, 000 dilution, shown in red. In Western blot, ab314504 was shown to bind specifically to SQSTM1 / p62. Target of interest was observed at 62 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in SQSTM1 knockout cell line (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (ab314504) at 1/1000 dilution

Lane 1:

Wild type HAP1 (human chronic myelogenous leukemia cell) whole cell lysate at 20 µg

Lane 2:

SQSTM1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 62 kDa

false

• WB

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Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lanes 1-4 : 15 seconds；Lanes 5-8 : 103 seconds

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (ab314504) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 5:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 6:

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

Lane 7:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 8:

NIH/3T3 (mouse embryonic fibroblast) 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: 62 kDa

false

• WB

Supplier Data

Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (AB314504)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lane 1 : 103 seconds; Lane 2 and 3 : 125 seconds

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [RM1079] - Autophagosome Marker (ab314504) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Rat spleen tissue 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: 62 kDa

false