Anti-LAMP2A antibody - Lysosome Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

IHC image of Lamp2A staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18528, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

ab18528 staining LAMP2 in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18528 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

ab18528 staining Lamp2A in CaCO2 cells treated with SB 202190 (ab120638), by ICC/IF. Increase of Lamp2A expression correlates with increased concentration of SB 202190, as described in literature.
The cells were incubated at 37°C for 3 hours in media containing different concentrations of ab120638 (SB 202190) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab18528 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

Side-by-side comparison of ICC performance using the rabbit polyclonal ab18528 and RabMab® ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab18528 or ab125068 overnight at +4°C at 3 different concentrations : 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® ab125068 shows negligiblenon-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 135 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

Side-by-side comparison of ICC performance using the rabbit polyclonal ab18528 and RabMab® ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab18528 or ab125068 overnight at +4°C at 3 different concentrations : 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® ab125068 shows negligible non-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 180 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling LAMP2A with ab18528 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab18528 Anti-LAMP2A antibody 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).

• WB

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Western blot - Anti-LAMP2A antibody - Lysosome Marker (AB18528)

Abcam recommends using 3% milk as the blocking agent.

Lanes 3 and 4 are human liver tissue lysates, total protein.

All lanes:

Western blot - Anti-LAMP2A antibody - Lysosome Marker (ab18528) at 1 µg/mL

Lane 1:

MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg

Lane 2:

Lung (Mouse) Tissue Lysate at 20 µg

Lane 3:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 20 µg

Lane 4:

Human liver left lobe tissue lysate - membrane extract (<a href='/en-us/products/unavailable/human-liver-left-lobe-tissue-lysate-membrane-extract-ab29086'>ab29086</a>) 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 1/10000 dilution

Predicted band size: 45 kDa

Observed band size: 105 kDa,30 kDa,35 kDa,55 kDa

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Exposure time: 8min