1. Coat Coverslips with Antibody

1. Prewarm (at 37 °C) 30 ml of RPMI 10% FCS media and 1 ml of BD Cytofix/Cytoperm.
2. Prepare a solution of 5 μg/ml of purified antibody in phosphate buffered saline (PBS). For activation of the NK92 cell line, use of anti-CD18  and anti-NKp30 is recommended.
   1. Mark one approximately dime sized circle for each condition on a #1.5 coverslip using a PAP pen. For a dual color experiment, there should be four conditions: unstained, dual stained, and two single stained conditions. Dispense 200 µl of antibody solution in each region and incubate at 37 °C for 30 min.
   2. Wash coverslips by gently immersing each one in 50 ml of PBS in a 50 ml conical tube at room temperature. Washing should occur immediately prior to the addition of cells and care should be taken to avoid antibody drying on the coverslip.

2. Activate NK Cells on Coverslips; Fix and Permeabilize

1. Isolate 5 x 10⁵ NK92 cells per condition. Centrifuge and decant supernatant. Wash once with 10 ml prewarmed media from step 1.1. Centrifuge and decant supernatant.
   1. Resuspend cells in prewarmed media from step 1.1 at a concentration of 2.5 x 10⁶/ml.
   2. Gently decant 200 µl to the center of the region created in section 1.2.1. Incubate at 37 °C for 20 min at 5% CO₂. (Note: this time can be extended or decreased depending on the biological function of interest. For NK cell granule polarization, 20 min is sufficient).
2. Following incubation of cells, gently wash coverslips by immersing each in 50 ml of room temperature PBS in a 50 ml conical tube.
3. Add 1 µl of Triton X-100 to 1 ml of prewarmed Fix/Perm solution from step 1.1 and vortex thoroughly. Fix and permeabilize by adding 200 µl of Fix/Perm buffer (step 2.3) to cells. Incubate for 10 min in the dark at room temperature.

3. Stain Cells

1. Prepare staining buffer: Phosphate buffered saline (PBS), 1% BSA, 0.1% Saponin.
   1. Prepare solution of primary antibody in 200 µl staining buffer (see step 3.1). (Note: antibody should be titrated prior to use). Avoid the use of primary antibody that is raised in the same species used to coat the coverslip (step 1.2). Also avoid Strepatividin-biotin linkages for STED imaging.
   2. Following section 2.3.1, gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply antibody solution created in section 3.1.1. Incubate 30 min in the dark at room temperature. (Recommended: incubate coverslips in slide box with a moist paper towel to maintain humidity).
2. Prepare solution of secondary antibody in 200 µl staining buffer. Recommended fluorophores are Alexa Fluor 488, Pacific Orange, and V500. Generally, a 1:200 dilution is suitable for STED imaging.
   1. Gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply secondary antibody solution. Incubate 30 min in the dark at room temperature.
3. Repeat washing and staining for additional proteins of interest. If detecting F-actin with Phalloidin, this can be included with secondary antibody, generally at a 1:200 dilution.

4. Mount Coverslips on Slides

1. Prepare mounting medium. Note: Prolong or Prolong Gold are preferable. VECTASHIELD must be avoided, as it is not compatible with STED. 2,2-thiodioethanol must be avoided if Phalloidin is used. Mowiol is acceptable.
2. Place approximately 10-20 µl of mounting medium on a slide. Invert coverslip (cell-side down) and mount coverslip gently, taking care to avoid introduction of air bubbles. Incubate slides for 24 hr (coverslip up) prior to imaging.
3. Seal edges of coverslip with nail polish.

5. Experimental Setup

1. Initiate required lasers and software. Initiate STED depletion laser at 100% power. Align STED laser, which in the case of commercial systems is often an automated procedure.
2. Focus the sample, beginning with single stained control, on the microscope using eyepieces.

6. Optimization of Settings

1. Scan the first channel and optimize laser power, excitation beam position and detector range. If possible, avoid a gain of >100. Capture the image in confocal to optimize settings. Line and/or frame averaging will increase resolution. Check for pixel saturation. Note: Some saturation is acceptable in confocal as application of STED will reduce the intensity of emission. For STED, an optimal pixel size will be below 30 nm however better resolution will be obtained with smaller pixel sizes. The size of the region of interest being imaged will dictate the lower limit of pixel size. Smaller pixel sizes may increase photobleaching.
   1. Apply STED depletion beam and capture image, starting with 50% depletion laser power. If an improvement in resolution is seen, more depletion laser power can be applied. At this stage, it may be necessary to adjust excitation laser power, line average, and/or gain.
   2. Apply time gating to reduce background (minimum 0.3 nsec). Adjust settings until an improvement in resolution over confocal can be seen. Resolution can be approximated by estimating full width half maximum (FWHM).  This represents the distance at the half maximal intensity of a Gaussian peak created by drawing a line profile across the structure of interest, and is a widely used method of estimating resolution.
   3. Once the first channel is satisfactory, initiate a second sequence for sequential scanning. In general, it is best to scan the longer wavelength fluorophore first. Repeat step 6.1 on second channel.
2. Confirm lack of spectral overlap by imaging single stained controls with both scan sequences. Mild spectral overlap can be corrected using spectral un-mixing features in the software, however should be avoided wherever possible.

7. Image Acquisition

1. Acquire images. For quantitative imaging, it is recommended to obtain at least 20 images/condition.  The exact number, however, should be defined according to the experimental question in concert with a statistical approach such as sample size calculation. Save experiment.

8. Deconvolution

1. Open file with deconvolution software or batch processor. Check parameters for each channel using software. Confirm each channel's excitation and emission spectra, STED depletion emission, and imaging direction (up or down, if the image is 3-dimensional) in particular.
2. Deconvolve using the deconvolution wizard. Default settings are generally adequate, however signal to noise ratio (SNTR) will vary from fluorophore to fluorophore and will need to be determined for each channel and each experiment individually.