Flow Cytometry-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 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 Jurkat cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Immunocytochemistry/ Immunofluorescence - 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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-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.

Immunocytochemistry/ Immunofluorescence - 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.

Immunocytochemistry/ Immunofluorescence - 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 25ºC and then incubated with ab25630 for 2 hours at 25ºC. 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

Immunohistochemistry (Frozen sections) - LAMP1 antibody [H4A3] - Lysosome Marker (ab25630)

IHC-Fr image of human brain section stained with ab25630. The brain sections were incubated in 10% normal donkey serum in 0.1% PBS- and 0.3x triton100 for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab25630, 1µg/ml) overnight at +4ºC. The secondary antibody was Alexa Fluor®568 donkey anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Ruma Raha-Chowdhury, University Of Cambridge, United Kingdom

Western blot - LAMP1 antibody [H4A3] - Lysosome Marker (ab25630)

All lanes : 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

developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 45 kDa
Observed band size : 48,120 kDa (why is the actual band size different from the predicted?)
Additional bands at : 120 kDa (possible glycosylated form).

Exposure time : 5 minutes

Ruma Raha-Chowdhury, University Of Cambridge, United Kingdom