A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Our research examines the epigenetic composition of chromatin packing domains, and how regulatory factors shape their structure and function. This protocol is most useful for investigating spatial relationships between targets and dense cellular environments, such as the nucleus. To begin, weigh bovine serum albumin, so that the final concentration in the required buffer volume for the experiment is 3%Add the bovine serum albumin to a centrifuge tube.

Tilt the centrifuge tube to a 45-degree angle to spread out the bovine serum albumin crystals, then add PBS to the tube. This prevents clumping of the crystals. Allow all bovine serum albumin crystals to dissolve naturally at room temperature and avoid shaking or vortexing.

Once the crystals are fully dissolved, add Triton X-100 to achieve a final concentration of 0.2%If crystals remain, pipette the solution up and down several times to mix the solution slowly without forming bubbles. Take the live cells from the incubator. Remove the cell culture medium from the dish.

Wash the cells by adding enough PBS to cover the cells, and then pipette out the PBS. Next, pipette enough fixative solution to cover the cells and leave the cells to fix for 10 minutes. Using a balance, weigh sodium borohydride to prepare a 0.1%quenching solution.

Add the sodium borohydride to a centrifuge tube. Pipette out the fixative solution from the dish. Then, add enough PBS to the dish to cover the cell surface.

Place the dish on a shaker for five minutes to wash the cells. Next, add the required volume of PBS to the sodium borohydride in the centrifuge tube. Vortex the tube to mix the quenching solution.

Remove the dish from the shaker and discard the PBS from the dish. Add enough quenching solution to cover the surface of the dish. Then, place the dish on a shaker for seven minutes to quench autofluorescence in the cells.

Discard the remaining quenching solution in the centrifuge tube into the appropriately-labeled liquid chemical waste container. Remove the dish from the shaker and then remove the quenching solution from the dish. Then, add enough PBS to the dish to cover the surface.

Place the dish on a shaker for five minutes to wash the cells. After incubation, remove the PBS and repeat the wash with PBS two additional times. Now, add enough blocking buffer to the dish to cover the surface.

Incubate the dish on a shaker for at least one hour to permeabilize the cell membranes and block binding sites. To prepare the primary antibody staining solution, transfer the required volume of the blocking buffer stock to a new centrifuge tube. Add the required volume of primary antibody stock to the blocking buffer to achieve the final concentration specified by the manufacturer.

Next, replace the blocking buffer with enough primary antibody staining solution to cover the surface of the dish and incubate on a shaker for one to two hours or for an overnight period. After primary antibody incubation, replace the primary antibody solution with washing buffer. Then, place the dish on a shaker for five minutes to wash the cells.

Repeat the washing buffer washed two additional times for a total of three washes. Prepare the secondary antibody staining solution according to the recommended concentration. Calculate the total staining solution volume by adding 0.5 milliliters to the volume required to cover the cells.

Transfer the calculated volume from the blocking buffer into a new centrifuge tube to prepare the staining solution. Then, add the appropriate volume of secondary antibody stock to the blocking buffer to obtain the correct final concentration. Wrap the centrifuge tube containing the secondary antibody staining solution with aluminum foil, and mix the solution by pipetting up and down.

Add enough secondary antibody staining solution to the dish to cover the surface. Place the dish on a shaker for 40 minutes to attach fluorophores to the labeled cellular targets. Cover the dish with aluminum foil to prevent fluorophore bleaching.

After 40 minutes, perform two PBS washes as demonstrated earlier. If storage is preferred, add enough PBS to the dish to cover the cell surface before storage. Wrap the dish with parafilm to prevent liquid evaporation, and then with aluminum foil to prevent fluorophore bleaching.

Store the wrapped dish at four degrees Celsius until ready to image. The sequential staining protocol produced representative three-color chromatin dSTORM images for multiple cell lines, including BJ Fibroblast, HeLa, and MCF 10A cells. The analysis pipeline was evaluated using simulated data sets representing normal uniform toroidal and random spatial distributions anchored to heterochromatin cluster positions.

Distinct spatial organization patterns produced characteristic distance histogram profiles when localization coordinates were analyzed relative to heterochromatin centroids. Simulated spatially segregated markers in a normal choroidal configuration produced minimal joint density, resulting in a flat distribution profile. Simulated overlapping marker patterns in a normal random configuration produced decreasing joint density with increasing distance from reference points.

DB scan-based identification of heterochromatin domains enabled categorization into small, medium, and large domains based on effective radius. Quantitative distance analysis showed that RNA polymerase II and H3K27ac localized near heterochromatin boundaries across small, medium, and large domains. Joint density analysis showed peak H3K27ac and RNA polymerase II colocalization just outside heterochromatin cluster boundaries.

Using this protocol, researchers can interrogate the seemingly disordered organization of chromatin to explore the relationship between its structure, regulatory elements, and functional outcomes. Following this procedure, various image-based or point cloud computational analyses can extract quantitative structural features from spatial data, depending on the imaging modality used. Researchers can extend this labeling method by optimizing additional markers and leveraging multi-channel data to enable more advanced and comprehensive analyses.