Immunocytochemistry/ Immunofluorescence - LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

ab25631 at 0.5µg/ml staining human HEK cells by immunocytochemistry. The cells were fixed with paraformaldehyde and incubated with the antibody for 1 hour. Bound antibody was detected using a goat anti-mouse IgG Alexa-Fluor ® 568. In the confocal image ab25631 labelling in red shows a distribution consistent with the location of lysosomes. Blue nuclear counterstain is present.

This image is courtesy of an Abreview submitted by Randal Moldrich on 31 March 2006.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-LAMP2 antibody [H4B4] - Lysosome Marker(ab25631)

Ab25631 staining human normal placenta. Staining is localized to the cytoplasm.
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.

Immunocytochemistry/ Immunofluorescence - LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

ab25631 staining LAMP2 in human THP-1 cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocking with 1% BSA at 22⁰C for 2 hours was done. Samples were incubated with primary antibody (1/400: in PBST and 1% BSA) for 16 hour at 4⁰C. An Alexa Fluor ® 568-conjugated goat polyclonal to mouse IgG was used at dilution at 1/500 as secondary antibody. Merge figure shows the bright field image and fluorescent image combined.

This image is a courtesy of Anonymous Abreview.

Western blot - LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631) at 1/500 dilution + whole cell lysate prepared from THP-1 macrophages at 30 µg

Secondary
Goat anti-mouse IgG conjugated to HRP at 1/2000 dilution
developed using the ECL technique

Observed band size : 110 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds

Primary antibody incubated for 12 hours at 4°C.
Gel running conditions: 12%
Blocked with 5% milk for 1 hour at 25°C.

This image was kindly supplied by Daniel Rodriguez by Abreview

Flow Cytometry-LAMP2 antibody [H4B4] - Lysosome Marker(ab25631)

Overlay histogram showing THP1 cells stained with ab25631 (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 (ab25631, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 THP1 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.