Alexa fluor 405 carboxylic acid succinimidyl ester

For STORM imaging, the photo-switchable secondary antibody consisting of a dye activator/reporter was custom prepared following the STORM-protocol sample preparation [53 (link)].
Briefly, secondary antibody used was a donkey anti-rabbit from Jackson ImmunoResearch Europe. The dyes were purchased as NHS ester derivatives: Alexa Fluor 405 carboxylic acid succinimidyl ester (Invitrogen), and Alexa Fluor 647 carboxylic acid succinimidyl ester (Invitrogen). Antibody labeling reaction was performed by incubating, for 40 min at RT, a mixture containing the secondary antibody, NaHCO3, and the appropriate pair of activator/reporter dyes diluted in dimethyl sulfoxide, anhydrous (DMSO) (Sigma-Aldrich).
Purification of labeled antibody was performed using NAP5 Columns (GE HealthCare).

Rabbit polyclonal anti-H2A (Abcam ab18255) and donkey-anti rabbit (711-005-152; Jackson ImmunoResearch) secondary antibodies were labeled in-house with different combinations of pairs of activator/reporter dyes (14 (link)). Briefly, dyes were purchased as NHS ester derivatives: Alexa Fluor 405 Carboxylic Acid Succinimidyl Ester and Alexa Fluor 647 Carboxylic Acid Succinimidyl Ester (Invitrogen). Labeling reactions were performed by incubating at room temperature for 40 min a mixture containing the secondary antibody in 0.12 M NaHCO₃, and the appropriate pair of activator/reporter dyes diluted in DMSO. Purification of labeled antibodies was performed using NAP5 Columns (GE HealthCare). The dye to antibody ratio was quantified using Nanodrop and only antibodies with a composition of 3–4 Alexa Fluor 405 and 0.9-1.2 Alexa Fluor 647 per antibody molecule were used for imaging.
Extracellular vesicles in H2B-GFP BMDMs were analyzed by STORM with the same protocol without the permeabilization step to avoid excessive staining of the nucleus. The cell membranes were stained with 50 µg/ml Concanavalin A-TRITC (Molecular Probes, Eugene, OR, USA).

Brefeldin A (BFA), from Sigma-Aldrich, was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) as a 10 mg/ml stock solution, and used at 5 µg/ml concentration. N-Hexanoyl-d-erythro-sphingosine (d-cer-C6), from Matreya, was dissolved in pure ethanol (Merck) as a 10-mM stock solution, and used at 20 µM concentration. Cycloheximide was purchased from A.G. Scientific, diluted to 1 M in DMSO as a stock solution, and used at 100 µM concentration. Sheep anti-human TGN46 was from AbD Serotec. Mouse monoclonal against HA was from Biolegend. Mouse monoclonal against Myc was from Sigma-Aldrich. Rabbit polyclonal antibody against Flag was from Sigma-Aldrich. Rabbit polyclonal antibody against GFP was from Abcam. Alexa Fluor-labeled secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies from Sigma-Aldrich. For STORM, the activator/reporter dye-conjugated secondary antibodies were in-house-labeled donkey-anti-mouse and donkey-anti-rabbit obtained from ImmunoResearch, used at a final concentration of 20 μg/ml. The dyes used for labeling were NHS ester derivatives: Alexa Fluor 405 Carboxylic Acid Succinimidyl Ester (Invitrogen), Cy3 mono-Reactive Dye Pack (GE HealthCare), and Alexa Fluor 647 Carboxylic Acid succinimidyl Ester (Invitrogen). Antibody labeling reactions were performed as previously reported (Borgman et al., 2020 (link)).