Hi John,

Have you tried FACS? You can isolate and purify exosomes of interest and then adsorb them to latex beads which can subsequently be analyzed by FACS.

If you think this is something you might want to try I will go dig up the paper/protocol I followed.

Hi John, it depends on your purpose. you can try with classical immunofluorescence using primary and secondary antibodies. Personally I use the primary mouse anti human LAMP1 (BD, code 555789) with the following protocols:

Fix cells with 70% ethanol in 96-well plates, permeabilise and block with a solution of 10% goat serum and 0.1% Tween 20 (Sigma-Aldrich) for 20 minutes at RT, and then stain with anti-lysosome-associated membrane protein-1 (LAMP-1) for two hours at RT. After washing, incubate cells with FITC-conjugated goat anti-mouse antibody for one hour in the dark at RT and, after further washing, add DAPI for nuclear staining.

You can also use fluorescent dyes like lysotracker that accumulate in lysosome and directly analyse cells.

Hi John,

we routinely perform cathepsin B immunofluorescence to visualize lysosomes, with the following protocol:

- Wash cells with PBS (x 3)

- Fix cells with 4% paraformaldehyde in PBS + 0.19% picric acid* for 20 min @ 37°C

- Wash with PBS (x 3)

- Permeabilize cells with fixative + 0.0125% (w/v) CHAPS for 10 min @ 37°C

- Wash with PBS (x 3)

- Block in PBS containing 5% FBS + 5% glycerol for 1 hr @ 37°C

- Primary Ab (mouse anti-cathepsin B [CA10], 100 ng/uL; Calbiochem #1M27L; 1:200) in blocking buffer, O/N @  4°C

- Wash with PBS (x 3)

- Secondary Ab (Alexa Fluor 488-conjugated goat anti-mouse; 1:1000) in blocking buffer 1 hr @ 37°C

- Wash with PBS (x 3)

- Wash with ddH2O (x 1)

- Mount in ProLong Gold with DAPI (InVitrogen) when still wet

As Federico Colombo already suggested, using lysotracker or lysosensor probes is also a good option, but be aware that some of them are not retained after fixation, therefore can only be used in live cells.

Frederico and Maria, thank you for your recommendations and protocols. I'll have to modify my own. I was fixing with PFA/MtOH and permeabilizing with saponin. I have not had much luck with our CatB antibody but thanks for the suggestion. Can't wait to try it!

Yes, I have used lysotrackers, rh-dextran, acridine orange, and neutral red already. I'd like something more specific for lysosomes and not endosomes or autophagosomes that can be fixed, so IF seems like the best bet here!

Hi John,

Besides Lyso Traker and LAMP antibodies, we also wanted  a more specific probe just for lysosomes. Thats when we explored the probe green DQ-BSA (from Invitrogen), a green self-quenched BODIPY® Dye Conjugate of Bovine Serum Albumin (also available in red). This conjugate does not fluoresce (because it is self- quenched), but becomes brightly fluorescent when reaching proteases at lysosomes. After DQ-BSA treatment, lysosomes can be easily observed by their bright green fluorescence. The protocol we used can be found in the following article:

Cell Microbiol. 2014 Apr;16(4):535-47. doi: 10.1111/cmi.12237.

A good Cathepsin antibody, as suggested by Maria Eugenia, is also a good option. Wishing you the best!