Anti-LAMP1 antibody [EPR21026] - Lysosome Marker

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunofluorescent analysis of 100% methanol-fixed Neuro-2a (mouse neuroblastoma cell line) cells labeling LAMP1 with ab208943 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Neuro-2a cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889), at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse spleen labelling LAMP1 with ab208943 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB IHC Kit with anti-rabbit HQ, anti-HQ HRP detection. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-LAMP1 antibody [EPR21026] ab208943 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling LAMP1 with ab208943 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular cytoplasmic staining on mouse spleen was observed, performed on a Leica Biosystems BOND^® RX instrument (PMID : 22008915). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Note : BOND^® is a registered trademark of Leica Biosystems Melbourne Pty Ltd.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse kidney tissue labeling LAMP1 with ab208943 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Cytoplasmic staining in the endothelial cells of glomeruli and epithelial cells of renal tubules (PMID : 23229015; PMID : 23635510). The nuclear counter stain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Flow cytometry overlay histogram showing NIH3T3 cells stained with ab208943 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab208943) (1x 10⁶in 100μl at 0.2μg/ml (1/11000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in NIH3T3 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunofluorescent analysis of 100% methanol-fixed NIH/3T3 (mouse embryo fibroblast cell line) cells labeling LAMP1 with ab208943 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889), at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methonol-permeabilized Neuro-2a (mouse neuroblastoma cell line) cell line labeling LAMP1 with ab208943 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling LAMP1 with ab208943 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular cytoplasmic staining on mouse spleen was observed, performed on a Leica Biosystems BOND^® RX instrument (PMID : 22008915). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Note : BOND^® is a registered trademark of Leica Biosystems Melbourne Pty Ltd.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

LAMP1 was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab208943 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab208943 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10 μg (Input).

Lane 2 : ab208943 IP in NIH/3T3 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab208943 in NIH/3T3 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (ab208943)

Predicted band size: 45 kDa

Observed band size: 90-120 kDa

true

• WB

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Western blot - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Exposure time : Lanes 1-2 : 3 minutes; Lane 3 : 5 seconds; Lane 4 : 10 seconds.

Blocking and dilution buffer : 5% NFDM/TBST.

The varying band sizes are due to different levels of glycosylation (PMID : 10212251, PMID : 26246576).

All lanes:

Western blot - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (ab208943) at 1/2000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma cell line) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 3:

Mouse kidney lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 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

Predicted band size: 45 kDa

Observed band size: 90-120 kDa

true

• WB

Supplier Data

Western blot - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (AB208943)

Exposure time : Lanes 1-2 : 15 seconds; Lane 3 : 5 seconds.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LAMP1 antibody [EPR21026] - Lysosome Marker (ab208943) at 1/1000 dilution

Lane 1:

Mouse lung lysate at 10 µg

Lane 2:

Mouse colon lysate at 10 µg

Lane 3:

Mouse liver lysate at 10 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 1/50000 dilution

Predicted band size: 45 kDa

Observed band size: 90-120 kDa

true