Click labeling and sample preparation for microscopy

A robust, basic protocol for alkyne lipid imaging in fixed cells has been described before (18). Based on the published protocol we have now developed three optimized protocol variants to enhance the sensitivity of alkyne lipid microscopy (variant A), and/or to combine imaging of alkyne lipids and fluorescent fusion proteins (variant B), or to allow for parallel protein detection by immunocytochemistry (variant C).

Cells were grown on glass coverslips and incubated with medium containing FCS and 10 or 2.5 μM alkyne lipid for 16 h and subsequently washed with 1% delipidated BSA in PBS, and with PBS alone. Cells were fixed in 3.7% formalin in PBS for at least 16 h and washed twice with PBS. Generally, all wash steps of fixed cells were performed for 10 min while gently agitating the samples. After washing once with 500 mM Tris/HCl, pH 7.4 and twice with click buffer (100 mM HEPES/KOH, pH 7.4), the samples were placed in a water bath at 43°C. The buffer was removed and 1 ml of prewarmed click buffer containing the azide detection reagent (generally at 10 μM concentration, but see figure legends for occasional exceptions) was added, followed instantly by the injection of 20 μl of 10 mM tetrakis(acetonitrile)copper(I) fluoroborate (CuTFB) in acetonitrile into the reagent solution (final concentrations: 200 μM CuTFB and 2% acetonitrile in the reaction mix) and brief agitation. After 15 min incubation, another 10 μl of copper catalyst solution was added. After another 15 min, the reagent solution was removed and samples were washed with click buffer two times. Samples probed with biotinylated azide reagents were washed sequentially with PBS and blocking buffer (PBS containing 2% BSA), and then incubated upside down on parafilm in a 30 μl drop of 1:120 streptavidin-Alexa488 (Dianova) in blocking buffer for 60 min, followed by two wash steps with blocking buffer. These steps were left out for samples probed with fluorescent azide reporters. Finally, all samples were washed with PBS six times and once with water before mounting in Mowiol 4-88 (Calbiochem).

Cells were transfected with vectors containing cDNA coding for fluorescent proteins using Lipofectamine2000 (Invitrogen) according to the manufacturers protocol. After 16 h, cells were incubated for 2 h with 1 μM (2S,3R)-2-aminooctadec-17-yn-1,3-diol (alkyne-sphinganine) added to the growth medium from ethanolic stock solutions. Cells were fixed by adding 1 ml of 3.7% formalin in click buffer and incubation for at least 16 h. Samples were washed sequentially with click buffer, 155 mM ammonium acetate, and click buffer before being placed in a water bath at 43°C. The buffer was removed and 1 ml of prewarmed click buffer containing 10 µM ApicSCy5 or ASBDP was added per well. Instantly, the reaction was started by addition of 20 µl of 10 mM or 100 mM CuTFB in acetonitrile (final concentrations: 200 μM or 2 mM, respectively), followed by brief agitation. After 15 min incubation, another 10 μl of copper catalyst solution was added. After a total of 30 min, the reagent solution was removed and the samples were washed with click buffer six times. Samples were mounted in Mowiol 4-88.

Cells were grown on glass coverslips and incubated with medium containing FCS and 1 µM nonadec-9-cis-en-18-ynoic acid (alkyne-oleate) for 24 h and washed with PBS. Samples were fixed in 3.7% formalin in PBS for 10 min and washed sequentially with PBS, 155 mM ammonium acetate, and PBS.

For immunofluorescence, antibody staining was performed before the click reaction. In detail, cells were permeabilized for 15 min in permeabilization buffer A [PBS/1% cold fish gelantine/0.01% saponin (Applichem #A2542)] for permeabilization of plasma membrane only or permeabilization buffer B (PBS/1% cold fish gelantine/0.1% saponin) for permeabilization of all cellular membranes. Cells were incubated with the primary and secondary antibodies in the corresponding permeabilization buffer for 1 h. Before the click reaction, cells were washed with click buffer before placing in a water bath at 43°C. The buffer was removed and 1 ml of prewarmed click buffer containing 10 µM ASBDP was added per well. Instantly, the reaction was started by addition of 20 µl of 100 mM CuTFB in acetonitrile (final concentration 2 mM) and brief agitation. After 1 h incubation, the reagent solution was removed and the samples were washed sequentially with click buffer, 20 mM EDTA, 155 mM ammonium acetate, and three times with PBS. Coverslips were mounted in Mowiol 4-88.

Mitotracker CMXRos (Molecular Probes #M7512) staining was performed according to the manufacturer’s instructions before the fixation and click reaction. In brief, living cells were incubated with 50 nM Mitotracker probe in growth medium for 15 min before washing and fixation as above. Before the click reaction, cells were washed with click buffer before placing in a water bath at 43°C. The buffer was removed and 1 ml of prewarmed click buffer containing 10 µM ASBDP was added per well. Instantly, the reaction was started by addition of 20 µl of 100 mM CuTFB in acetonitrile (final concentration 2 mM), followed by brief agitation. After 1 h incubation, the reagent solution was removed and the samples were washed sequentially with click buffer, 20 mM EDTA, 155 mM ammonium acetate, and three times with PBS. Coverslips were mounted in Mowiol 4-88.

Staining of cellular nuclei by 4’,6-diamidino-2-phenylindole (DAPI) and lipid droplets by 4,4-difluoro-2.3,5.6-bis-tetramethylene-4-bora-3a,4a-diaza-s-indacene (LD540) (33) was performed directly before mounting.