Anti-LAMP1 antibody [H4A3] - Lysosome Marker

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SCARB2 KO MCF7 (SCARB2 knockout human breast adenocarcinoma epithelial cell) cells labelling LIMPII with ab314217 at 1/50 (10.54 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 no staining in SCARB2 KO MCF7 cells, and lysosomal staining in parental MCF7 cells.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab25630 Anti-LAMP1 mouse monoclonal antibody was used to counterstain tubulin at 1/200 0.2ug/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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized IMR-90 (human lung fibroblast) cells labelling IDUA with ab323701 at 1/50 (10.2 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 lysosome staining in IMR-90 cells(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : JAR.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab25630 Anti-LAMP1 mouse monoclonal antibody 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 2ug/ml dilution.

Counter stain secondary antibody only control : Secondary antibody is ab150115 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 2ug/ml dilution.

• ICC/IF

AbReview15890****

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

ab25630 at 1/500 dilution staining LAMP1 in human gastric cancer cells by immunocytochemistry/ immunofluorescence. Sections were methanol fixed, permeabilized in 0.5% Triton X-100 prior to blocking in 10% NGS/1% BSA for 1 hour at 25°C and then incubated with ab25630 for 2 hours at 25°C. Alexa fluor® 594 mouse polyclonal to mouse Ig, diluted 1/600, was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• ICC/IF

AbReview5778****

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

ab25630 at 1/100 staining human Primary Gingival epithelial cells by ICC/IF. The cells were ixed with 4% paraformaldehyde, permeabilized with 0.5% saponin and then blocked overnight with 10% goat serum, 5% BSA. The cells were incubated with the antibody for 1 hour and then a FITC conjugated goat polyclonal antibody was used as the secondary. The cells were counterstaind with DAPI for the nucleus and Cell Tracker Blue for the cytoplasm.

This image is courtesy of an anonymous Abreview

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

ab25630 staining Human normal placenta. Staining is localized to the cell membrane.
Left panel : with primary antibody at 1 ug/ml. Right panel : isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

Overlay histogram showing Jurkat cells stained with ab25630 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab25630, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

ab25630 stained Hela cells. The cells were 100% methanol fixed for 5 minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with ab25630 at 5μg/ml and ab202272 (Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594) – pseudo-colored red) overnight at +4°C. The secondary antibody (pseudo-colored green) was a Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) used at a 1/1000 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

IHC image of LAMP1 staining in human placenta 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 ab25630, 1μg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

ab25630 staining LAMP1 in human HaCaT keratinocytes by Immunocytochemistry/ Immunofluorescence. Cells were fixed with acetone, permeabilized with ice cold acetone and blocking with 4% PBS, 0.4% BSA and goat serum was performed for 16 hours at 4⁰C. Samples were incubated with primary antibody (1/100 : in 10% blocking solution in PBS) for 1 hour at 23⁰C. An Alexa Fluor ® 680-conjugated goat polyclonal to mouse IgG was used at dilution at 1/200 as secondary antibody. Alexafluor-680 signal is pseudocolored green in the image.

This image is a courtesy of Anonymous Abreview. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling LAMP1 with ab278043 at 1/100 (5.45 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (green). Confocal image showing cytoplasmic staining in HeLa. ab25630 Anti-LAMP1 mouse monoclonal antibody was used to counterstain at 1/500 (red). The Nuclear counterstain was DAPI (blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution

• WB

Lab

Western blot - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab25630 (1/1000) overnight at 4°C. Antibody binding was detected using ab9485 (rabbit anti-GAPDH); at a 1/10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (ab25630) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

Jurkat membrane lysate at 20 µg

Lane 3:

MCF-7 cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 115-120 kDa

false

• WB

AbReview20161****

Western blot - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (AB25630)

WB image of LAMP1 (ab25630) on Mouse liver. Lanes were loaded 20 ug of Liver tissue lysate Lane 1. iron treated 3 month old liver, lane 2. untreated 3 month old liver, Lane 3. one month old untreated liver.

All lanes:

Western blot - Anti-LAMP1 antibody [H4A3] - Lysosome Marker (ab25630) at 1/10000 dilution

Lane 1:

Iron treated 3 month old liver at 20 µg

Lane 2:

Untreated 3 month old liver at 20 µg

Lane 3:

One month old untreated liver

Predicted band size: 45 kDa

Observed band size: 120 kDa,48 kDa

true

Exposure time: 5min

This image is courtesy of an abreview submitted by Ruma Raha-Chowdhury, University Of Cambridge, United Kingdom