ICC: Staining Protocol - mCLING

General considerations

For homogeneous labeling of the plasma membrane, make sure to dissociate cells before plating and use the cells at a confluence rate not above 50% to 70%. Make sure to work with healthy viable cells. mCLING can strongly bind to cell debris.

The coverslips must have the least interaction possible with mCLING molecules. This is particularly important when the cells directly imaged from the coverslip where they are cultured. mCLING is a positively charged molecule. Chemical procedures used to clean glass coverslips typically leave the surface negatively charged, which leads to binding of mCLING molecules and a strong background signal. Similarly, some extracellular matrix molecules used for coverslip coating, like collagen, fibronectin and laminin, have both positive and negative charges, resulting also in a strong background signal that makes it difficult to distinguish the borders of the labeled cells. A good solution to this problem is to use a positively charged coating like poly-L-lysine (PLL).

mCLING has to be stably fixed to the membranes of the stained cells. Especially for subsequent immunostaining, it is important to cross-link mCLING to membrane proteins, via amine-fixative reactions. mCLING offers seven amine groups for its cross-linking with aldehyde fixatives. However, paraformaldehyde does not provide sufficient fixation. Therefore, a mixture of 4% paraformaldehyde (PFA) and 0.2% glutaraldehyde is strongly advised. Unfortunately, antibody binding may be affected by this stronger fixation and a careful evaluation of the antibody used in this approach is mandatory.

Materials and reagents

Physiological buffered saline (e.g. Tyrode’s buffer for cultured neurons and Ringer’s buffer for cultured cell lines like COS-7)
mCLING lyophilized
PBS: Phosphate buffered saline, pH 7.4
Fixation buffer: 4% PFA, 0.2% glutaraldehyde in PBS
Ice
Quenching buffer: 100 mM NH₄Cl, 100 mM Glycine in PBS
Blocking buffer: 0.1% Triton X-100, 2.5% bovine serum albumin (BSA) in PBS
Mounting medium

Procedure for labeling membrane trafficking

Pre-warm the physiological buffered saline of choice to 37°C.
Prepare a working mCLING solution with a concentration between 1-0.2 nmol/ml, diluted in physiological saline. Keep the solution warm at 37°C. Cells cultured on feeder layers may require higher mCLING concentrations up to 0.8 µM. Never use mCling at a higher concentration as 2 µM since it has a toxic effect.
Remove culture medium and carefully wash the cells with pre-warmed physiological buffer, three times briefly.
Apply the mCLING solution. The labeling protocol will depend on the aim of the experiment, and on the cell type:

For endocytosis and membrane trafficking in stable cell lines: Incubate the cells with mCLING solution for 5 to 10 min at 37°C. This should allow the visualization of the plasma membrane and endocytosed organelles. Longer incubation periods (e.g. 20 min) might be necessary to see molecular recycling back to the plasma membrane. At this point, mCLING incubation can be combined with fluorescently labeled ligands for endocytosis, like transferrin, low-density lipoprotein (LDL) or epidermal growth factor (EGF).
Synaptic vesicle recycling during active release in neurons: Incubate the neurons with the mCLING solution for 1 min to ensure homogeneous plasma membrane labeling. Immediately after, apply the stimulation of your choice (e.g. electrical stimulation or high K⁺ solution). Be aware that mCLING is not a washable molecule, as it is the case for other endocytosis markers typically used to study synaptic vesicle recycling in neuronal preparations (e.g. FM 1-43 or FM 4-64). Thus, fluorescent signal coming from the plasma membrane cannot be removed by perfusing the cells with buffer.
Synaptic vesicle recycling during spontaneous release in neurons: Incubate the neurons in 500 μl physiological buffer containing 1 μM tetrodotoxin and mCLING for 15 min at 37°C.

Wash excess mCLING with physiological buffered saline.
Immediately place the coverslip in fixation solution. Incubate for 20 min on ice followed by an additional 20 min at room temperature.
Wash with PBS.
Incubate for 30 min in 2 ml quenching buffer.
Wash with PBS.
Block for 5 min in blocking buffer at room temperature. Repeat this step 3 times.
Incubate with primary antibody for 1 h at room temperature in blocking buffer. Note: Overnight incubation at 4°C is not recommended as it might affect the stability of mCLING on the labeled membranes, resulting in a loss of signal.
Wash 3 times 5 min each with PBS at room temperature.
Incubate with secondary antibody in blocking buffer for 1 h at room temperature.
Wash 3 times for 5 min each with PBS at room temperature.
Mount coverslips.

Procedure for labeling only the outer plasma membrane

Prepare 100 ml of physiological buffer pre-chilled to 4°C and 100 ml equilibrated to room temperature.
Prepare a multi-well plate with fixation buffer and place it on ice for 30 min.
Prepare a working mCLING solution with a concentration between 0.1 to 0.2 μM diluted in cold physiological buffered saline. Keep the solution on ice for 30 min.
Gradually reduce the temperature of the cells by dipping the coverslips once into the physiological buffered saline at room temperature, and twice into the cold physiological buffered saline. Immediately transfer the coverslips to the multi-well plate with pre-chilled physiological buffered saline and incubate the cells on ice for 5 min.
Remove the physiological buffered saline and immediately add the previously cooled mCLING solution and incubate for 10 to 15 min. Note: At low temperature mCLING requires longer incubation times to homogeneously reach the cell plasma membrane.
Remove the mCLING solution and briefly wash excess of mCLING two times with cold physiological saline.
Immediately transfer the coverslips into ice-cold fixation buffer and incubate for 20 min on ice followed by 20 min at room temperature.
Wash with PBS.
Incubate for 30 min in 2 ml quenching buffer.
Wash with PBS.
Block for 5 min in blocking buffer at room temperature. Repeat this step 3 times.
Incubate with primary antibody for 1 h at room temperature in blocking buffer. Note: Overnight incubation at 4°C is not recommended as it might affect the stability of mCLING on the labeled membranes, resulting in a loss of signal.
Wash 3 times 5 min each with PBS at room temperature.
Incubate with secondary antibody in blocking buffer for 1 h at room temperature.
Wash 3 times for 5 min each with PBS at room temperature.
Mount coverslips.

Note: For more background information and more application specific protocols, refer to Revelo NH and Rizzoli SO, 2016.