DiOlistics: Delivery of Fluorescent Dyes into Cells

Abstract

DiOlistic labeling utilizes a particle-mediated delivery system to incorporate dye into cells. Because of its random nature, this technique generates sparse fluorescent labeling which is well suited for the study of neuronal dendritic branching and dendritic spine morphology. DiOlistics is a quick, reliable and nontoxic method that can be used in combination with other techniques such as immunostaining, biolistic DNA transfection, and retrograde tracing. In this article, we describe the methods for diOlistic labeling of neurons from rodent brain slices using DiI and the imaging of neuronal and synaptic morphology using confocal microscopy.

1. Introduction

DiOlistics uses a gene gun to introduce fluorescent dyes into cells which then get incorporated into the cytoplasm or the plasma membrane. This technique most commonly uses DiI (1-1′-dioctade-cyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate), alipophilic dye that will partition in and diffuse through the cell membrane providing a well-defined outline of neuronal processes which is ideal for high resolution confocal imaging.

The primary application of this technique has been the study of neuronal morphology in the central nervous system, but it can be applied to a variety of cell types, from living or fixed tissues, as well as in diverse species such as rodents, primates, and zebrafish (1–3). Another advantage of diOlistics is that it can be used in animals of all ages, making it suitable for developmental studies, as well as a complement to behavioral assays in adult or aged animals. DiOlistics is a versatile labeling technique that can be used in combination with immunostaining, to identify subpopulations of cells (4–7). Additionally, the technique can be used in combination with biolistic particle-mediated DNA delivery, and/or retrograde tracing for circuit mapping or cell identification (2, 8, 9).

The procedures of the technique are described in detail in the following sections. First, trans-cardiac perfusion is used for efficient fixation of the brain and all other tissues. Fixed brains are then sliced into sections. Bullets are prepared by coating tungsten beads with DiI and a gene gun is used to shoot the fluorescent bullets onto the brain slices, sparsely labeling cells within minutes. Incorporating immunostaining is an optional step described here and recently, improvements have been suggested for the combination of the methods (9, 10). After mounting the sections, images of fluorescently labeled neurons are acquired using confocal microscopy for detailed analysis of dendrite and spine morphology.

2. Materials

Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents at room temperature, unless indicated otherwise. Follow all waste disposal regulations when discarding waste materials.

2.1. Bullet Materials

1. Tungsten Beads, 1.7 μm diameter (Bio-Rad, Hercules, CA, USA). To prepare aliquots, suspend 6 g tungsten beads into 2 mL methylene chloride. Vortex to create a homogenous solution and pipette 100 μL of bead solution into individual eppendorf tubes resulting in 300 mg aliquots. Store at room temperature until needed in step 3.1.1 (see ^{Note 1}).

2. DiI (Invitrogen, Carlsbad, CA, USA).

3. Tefzel bullet tubing (Bio-Rad).

4. Polyvinylpyrrolidone (PVP): Prepare 10 mL of 10 mg/mL PVP solution dissolved in deionized water.

5. Tubing Prep Station (Bio-Rad) connected to a nitrogen tank.

6. 12 mL Syringe with tubing adaptor, made of flexible tubing with slightly larger diameter than Teflez tubing.

7. Tubing cutter (Bio-Rad).

2.2. Perfusion Materials

1. Ketamine/xylazine anesthetic cocktail: 100 mg/mL ketamine and 20 mg/mL xylazine.

2. Butterfly needle with blunted tip. Use a Dremel-like tool to smooth the pointed tip of the needle.

3. Pump or gravity system used for perfusion.

4. Scissors.

5. Chilled container.

2.3. Solutions

1. Phosphate Buffer Solution (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄O, 2 mM KH₂PO₄O. To make 1 L, add 8 g NaCl, 200 mg KCl, 1.44 g Na₂HPO₄O, and 240 mg KH₂PO₄O to H2O. Use NaOH to adjust pH to 7.2–7.4.

2. Fixative Solution: 4% (w/v) paraformaldehyde and 4% (w/v) sucrose in PBS. Weigh paraformaldehyde under the chemical hood and mix ingredients in a large beaker. Heat and stir to solubilize, but do not boil. If needed, add NaOH pellets to solubilize and bring to pH neutral once in solution. Filter before using. Good for about 3 weeks if stored at 4°C.

3. Sucrose solutions: 15% and 30% (w/v) sucrose in PBS.

4. Permeabilization solution: 0.01% (v/v) Triton X-100 in PBS.

5. Blocking solution: 10% (v/v) goat serum, 0.01% (v/v) Triton X-100 in PBS.

2.4. Shooting and Mounting Materials

1. Vibratome.

2. 24-Well plate.

3. Paintbrush.

4. Gene Gun and accessories (Fig. 1): o-rings, barrel liner, diffusion screens, cartridge holders, cartridge extractor tool (Bio-Rad). Modify diffusion screens (see ^{Note 2}) by removing the radial poles with pliers as shown in Fig. 2.

5. Isopore membrane filter paper 3.0 μm pore size (Millipore, Billerica, MA, USA).

6. Helium gas tank.

7. ProLong Gold Antifade mounting medium (Invitrogen).

8. Slides: 25 mm × 75 mm × 1 mm and coverslips: 18 mm × 18 mm × 1 mm.

9. Nail polish.

Fig. 1.

Shooting Materials: (a) Gene gun, (b) Barrel liner, (c) O-ring, (d) Tubing cutter, (e) Modified diffusion screens, (f) Isopore membrane filter paper picture with box, (g) Teflez tubing, (h) Cartridge holder, (i) Cartridge extractor tool, (j) Scintillation vial, (k) Syringe with tubing adapter.

Fig. 2.

(a) Diffusion screens are modified by removing the radial poles with pliers. (b) A filter membrane should be placed between two diffusion screens and then inserted at the bottom of the barrel liner, twisting to ensure a secure fit. (c) When shooting, the barrel liner should be centered on the well and pressed flat onto the well plate.

2.5. Evaluation Materials

1. Rocking platform.

2. Primary and fluorescence-conjugated secondary antibody.

3. Confocal microscope equipped with a 561 nm laser (LSM 520 META, Zeiss, Germany).

3. Methods

3.1. Preparing DiI/Tungsten Bead Bullets

1. Resuspend a 300 mg tungsten aliquot in 300μL of methylene chloride. This amount will be enough for 3 sets of bullets. Mix well by sonicate for 3 min to disrupt any bead clumps.

2. Pipette 100 μl of the tungsten bead solution on glass side.

3. Combine 13.5 mg DiI with 450 μL of methylene chloride for a final concentration of 3 mg/100 μL.

4. Coat beads with dye by pipetting on top of the beads a 100 μl of DiI solution. Mix thoroughly using pipette tip until methylene chloride evaporates and beads turn light grey.

5. Place on a piece of wax-coated weigh paper under the glass slide and use a razor blade to scrape beads off the glass slide and dice into a fine powder.

6. Scrape beads onto weigh paper and funnel beads into a 15 mL conical tube.

7. Add 3 mL of water and sonicate in water bath for 10–30 min (see ^{Note 3}).

8. Cut Teflez tubing slightly longer than the length of the tubing prep station. Use a 12 mL syringe with tubing adaptor to pass the PVP solution through the Teflez tubing and then expel it out. Reuse solution to coat more tubing if several batches of bullets are prepared simultaneously and discard PVP solution after use.

9. Vortex the beads at a low speed to produce a homogenous solution and simultaneously use the syringe to pull the bead solution through the tubing so beads are evenly distributed throughout the tubing (see ^{Note 4}).

10. Feed the tubing through the prep station and wait about 1 min to allow the beads to settle. Use the syringe to gently remove the water so that only the beads remain (see ^{Note 5}).

11. Spin the tubing in the prep station and dry with nitrogen gas until water droplets are no longer visible, approximately 10–20 min.

12. Cut bullets with tubing cutter into 13 mm bullets and store in a container, such as a scintillation vial. Wrap vials in foil to protect from light. Bullets can be prepared in advanced and stored for several months.

3.2. Trans-Cardiac Fixation and Brain Extraction

This procedure is preferred when possible because it allows for reliable and efficient fixation. Alternatively, a block of fresh tissue can be fixed before or after slicing (3).

1. Anesthetize the mouse with an intra-peritoneal injection of ketamine/xylazine solution (0.1 mL/20 g mouse) (see ^{Note 6}).

2. Make a sagittal incision along the midline exposing the liver and the chest. Cut through the diaphragm and ribs, along the sternum toward the mouse’s left side. Clamp sternum with hemostats and flip above the mouse’s head to hold chest cavity open, exposing the heart.

3. Using a small scissor, make a small incision into the left ventricle and insert a butterfly needle with blunted tip (see ^{Note 7}).

4. Use a small scissor to cut the vein coming into the right atrium (see ^{Note 8}).

5. Perfuse 10–15 mL of cold PBS at a speed of 2.5 mL/min (see ^{Note 9}). A pump or gravity can be used in this step.

6. Perfuse about 20 mL of paraformaldehyde solution at a speed of 2.5 mL/min. Once the animal is stiff, stop perfusion (see ^{Note 10}).

7. Decapitate the mouse and gently remove the brain.

8. Place brain in chilled container (e.g., glass scintillation tube) with 10–20 mL 4% paraformaldehyde solution for 30 min (see ^{Note 11}) on ice.

9. Wash in PBS for 5 min on ice, three times.

10. Incubate brains for 10 min in 15% sucrose solution followed by 10 min in 30% sucrose solution to improve neuronal morphology (see ^{Note 12}).

11. Keep at 4°C in container filled with PBS until needed.

3.3. Cutting the Brain Slices

1. Glue the brain to a vibratome platform to allow brain sectioning at the desired orientation. The orientation should be determined based on the region of interest and the morphology of the cells.

2. Add cold PBS to cover the brain and keep solution and chamber on ice (see ^{Note 13}).

3. Cut sections of 200–300 μm thickness with a sharp blade.

4. Transfer slices with a brush to individual wells of a 24-well plate containing PBS (see ^{Note 14}).

3.4. DiI Staining

1. Remove PBS from wells and place slices in the center of the wells using a paintbrush.

2. Prepare gene gun: Load bullets into cartridge holder, assemble diffusion screens and filter paper into the barrel liner (Fig. 2) and insert both into gene gun (see ^{Note 15}).

3. Press the barrel liner flat onto the surface of the 24-well plate (Fig. 2) and shoot once in each well at 120–180 psi helium gas pressure (see ^{Note 16}).

4. Quickly wash slices with PBS, two times. For the third wash, cover slices with foil and place on a rocking platform for 30 min.

5. Mount slices: Gently place slice on glass slide and add 8–10 μL ProLong Gold Antifade mounting media on top avoiding air bubbles. Carefully place a coverslip over the slice and leave at room temperature overnight for mounting media to dry (see ^{Note 17}).

6. Seal edges of the coverslip with nail polish after mounting media has dried. Then, store slides at 4°C (see ^{Note 18}).

3.5. Immunostaining

Immunostaining is an optional addition to this method and, when used, it should be performed after DiI shooting and before mounting (see ^{Note 19}).

1. Permeabilization: Incubate slices in permeabilization solution (300–500 μL per well) for 15 min at room temperature (see ^{Note 20}).

2. Blocking: Incubate slices in blocking solution for 30 min at room temperature on a rocking platform.

3. Primary antibody: Dissolve primary antibody in blocking solution (e.g., 1:1,000) and incubate slices in 300–500 μL per well for 1–2 h on a rocking platform (see ^{Note 21}).

4. Wash slices with PBS for 5–15 min, three times on a rocking platform.

5. Secondary antibody: Dissolve fluorescence-conjugated secondary antibody in blocking solution (e.g., 1:1,000) and incubate slices in 300–500 μL per well for 30 min on a rocking platform.

6. Wash slices with PBS for 5 min, four times on a rocking platform.

7. Mount slices as described in the “DiI staining” section.

3.6. Confocal Microscopy

A confocal microscope equipped with a 561 nm laser (LSM 520 META, Zeiss, Germany) is used to visualize fluorescent labeling (Fig. 3).

Fig. 3.

(a) Confocal image of a labeled medium spiny neuron of the mouse nucleus accumbens obtained with a 20× objective. The high intensity of the fluorescent labeling and the low background is suitable for the study of dendritic branching and neuronal morphology. (b) Confocal image of a labeled tertiary dendrite obtained with a 63× objective, displaying the diverse morphology of dendritic spines.

1. Acquire images (512 × 512 pixels, 8 bit) by averaging two frames at a speed of 7 (scale 0–10).

2. Set pinhole size at 1Airy Unit (AU) for each objective. The size in microns will vary depending on the objective and wavelength.

3. Adjust the detector’s gain to achieve saturation only in the brightest pixels. In this case, the detector’s gain was set around 700. The use of digital gain and offsets is not recommended; increase laser power instead if needed.

4. To capture the entire cell morphology, acquire a z-stack of images with a 20× objective (air, NA= 0.8). Pinhole size is 2.0 μm and z-stack interval is set to 1.4 μm (0.7 times the pinhole size). Recommended laser power is 4–15%, but optimal power might vary depending on the microscope and the fluorescence intensity.

5. To image dendrites, a 63× objective (water, NA= 1.2) is recommended and a digital zoom of 2 can be added. Pinhole size of 1AU is 1.0 μm and z-stacks of images are collected at a 0.7 μm interval. Recommended laser power is 40% but optimal power might vary with the microscope and fluorescence intensity (see ^{Note 22}).