CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of the Transparent Mouse Skin Tissue

NOTE: All mice used in this study were on a C57BL/6 genetic background

1. Collection of mouse skin tissue.
   1. Humanely euthanize the mice by cervical dislocation.
   2. Carefully remove hairs from the relevant skin area with a trimmer taking care not to wound the skin.
   3. Wash the skin to decontaminate with 70% ethanol in Phosphate Buffered Saline (PBS).
   4. Lift dorsal neck skin with forceps and make an incision with scissors.
   5. Dissect a large area of dorsal mouse skin (approximately 1.5 x 4 cm).
   6. Flatten skin, dermis-side down, on a filter paper and make note of the anterior-posterior orientation of the sample.
   7. Trim filter paper around the dissected skin, and place in a 15 mL tube filled with freshly prepared 4% paraformaldehyde (PFA) solution in PBS.
   8. Fix for 1 hr at room temperature, or overnight in the refrigerator at 4 °C.
   9. Wash the skin 2x 5 min in PBS in a 15 mL tube.
      NOTE: The following (1.1.10 - 1.1.12) are optional steps for long term storage of tissue.
   10. Dehydrate dissected skin in PBS with increasing concentration of ethanol (25%, 50%, 70%) in a 15 mL tube during 1-hr wash steps at room temperature.
   11. Store dehydrated skin in 70% ethanol in PBS in a 15 mL tube at 4 °C until further use.
   12. Four hours before clearing, rehydrate skin tissue in PBS with decreasing concentration of ethanol (70%, 50%, 25%, 0%) in a 15 mL tube during 1-hr wash steps at room temperature.
2. Clearing the mouse skin biopsies
   1. Prepare the CUBIC1 clearing solution by dissolving 3.85 g urea and 3.85 g N,N,N',N'-tetrakis (2-hydroxypropyl) ethylenediamine in 5.38 mL distilled water on a heater set to 60-70 °C. Use a hot stirrer.
   2. Add 2.31 g polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 to the solution once it is clear and has cooled down to room temperature.
   3. Cut the mouse skin with a sharp razor blade into biopsies of approximate dimensions 0.2 x 0.5 cm, and submerge in 5 mL of CUBIC1 clearing solution in a 15 mL tube. To optimize the visualization of hair follicles, ensure that the longer side of the biopsy is cut along the antero-posterior direction of the sample.
   4. Place on a rotating platform in a hybridization oven at 37 °C.
   5. Change the clearing solution after 7 days. Prepare a fresh CUBIC1 solution prior to use.
   6. Check the transparency of the tissue after 7 days. If necessary, leave biopsies in CUBIC1 clearing solution until the tissue is completely transparent.
   7. Once the skin biopsy is transparent, remove the CUBIC1 solution, and add 4 mL of 1x PBS to wash the tissue 4 times for 6 hr at 37 °C.
   8. Wash the skin tissue in 20% w/v sucrose in PBS in a 15 ml tube for 4 hr at 37 °C.
   9. Freeze the tissue in mounting medium Optimal Cutting Temperature (OCT) Compound in a 15 mL tube overnight in a -80 °C freezer.
      NOTE: This step will increase the biopsy's permeability for antibody penetration in subsequent steps.

2. Immunofluorescence Staining

1. Thaw the tissue from (step 1.2.9) to room temperature for 2 - 3 hr.
2. Wash the tissue in the 15 mL tube with 5 mL of PBS for 8 hr at room temperature to remove OCT Compound.
3. Transfer the biopsies to a 2 mL tube, and incubate tissue in 1 mL rabbit anti-Keratin14 or rabbit anti-Ki67 antibody, both diluted 1:100 in PBST (PBS + 0.1% Triton-X100) for 3 days on a shaker in a 37 °C oven.
4. Transfer the biopsies to a 15 mL tube, and wash the tissue 4 times for 6 hr in 5 mL of PBST on a shaker in a 37 °C oven.
5. Transfer the biopsies to a 2 mL tube, add 1 mL anti-rabbit Alexa594 secondary antibody diluted 1:100 in PBST and incubate 3 days on a shaker in a 37 °C oven.
6. Transfer the biopsies to a 15 mL tube, and wash the tissue 4 times for 6 hr in 5mL of PBST on a shaker in a 37 °C oven.
7. Transfer the biopsies to a 2 mL tube, add 1 mL 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain solution (1:1,000) in PBST and incubate overnight on a shaker in a 37 °C oven.
8. Remove DAPI counterstaining solution, and add 1 mL of PBST to the 2 mL tube to wash tissue 4 times for 6 hr on a shaker in a 37 °C oven.
   NOTE: Optional step: Biopsies can be stored in the dark in 1x PBS with 0.02% sodium azide for at least 3 weeks.

3. Imaging

1. Prepare the CUBIC2 clearing solution, which contains 50% (w/v) sucrose, 25% (w/v) urea, 10% (w/v) 2,2′,2′'-nitrilotriethanol, and 0.1% (v/v) Triton X-100.
2. Incubate the skin tissue in 1 mL CUBIC2 solution in a 2 mL tube on a shaker for 24 hr in a 37 °C oven. This step will even the refractive index of the tissue.
3. Check the clarity of the tissue. Once it is clear, position the entire skin biopsy (0.2 x 0.5 cm) on its longer side onto a glass coverslip, such that the direction of the length of the hair follicles is parallel to the coverslip surface (#1 24 x 60 mm).
   NOTE: Optional step: Antibody-stained biopsies can be stored in CUBIC2 solution for about 7 days. Nile Red-stained biopsies can only be stored in CUBIC2 solution for up to 1 day, and should be imaged as soon as possible.
4. Prepare imaging chamber (Figure 1).
   ​NOTE: Consumables required for the preparation of the imaging chamber are: blue tack, play dough or similar, and 2 coverslips (24 x 50 mm) (Figure 1A).
   1. Prepare two thin strips of blue tack (diameter approximately 1 mm x 2 cm), and two coverslips (Figure 1B).
   2. Place blue tack strips on one cover slip, allowing enough space for skin biopsy (Figure 1C, step 3.4.3).
   3. Place skin biopsy in between strips on the coverslip in a drop of CUBIC2 solution (Figure 1C).
   4. Cover skin biopsy with the second coverslip (Figure 1D).
5. Place the imaging chamber with the mounted skin biopsy onto the stage of a confocal microscope and move the tissue into the light pathway.
6. Scan the sample using a mercury or halogen light source and standard epifluorescence filters (e.g., DAPI/GFP/CY3/CY5) to identify fluorescently stained regions of interest.
7. Image regions of interest using a 10X and 20X objective (NA 0.75) and standard confocal fluorescence imaging techniques (e.g., with DAPI stained samples illuminate with a 405 nm laser and collect fluorescence signal between 425-475 nm, and with Alexa Fluor 594 or Nile Red stained samples illuminate with a 561 nm laser and collect fluorescence signal between 570-620 nm).
8. Generate image Z-stacks of each region of interest using microscope Z-stack image acquisition software (e.g., using NIS elements imaging software 4.13 as described below).
   1. Ensure optimal laser power and select PMT HV/Offset settings to collect sample fluorescence.
   2. Open the "ND Acquisition" toolbar from "Acquisition Controls".
   3. Select the "Z series Setup" tab (ensure all other tabs are unselected).
   4. While live scanning, focus to the top of the sample and press the "Top" button.
   5. Focus on the bottom of the sample and press the "Bottom" button.
   6. Input required step size (or press optimized step size button).
   7. Press the "Run Now" button.
   8. Save resultant Z-stacks as individual Tiff image stacks (1 fluorochrome per image Z-stack).
9. Use image Z-stacks to reconstruct 3D volume regions of interest using 3D analysis software (e.g., using Imaris x64 7.2.3 as described below).
   1. Start analysis software.
   2. Choose the "Surpass" button in the toolbar for 3D volume generation.
   3. Open first color image Z-stack (e.g., DAPI).
   4. Use the 'add channel' tool to add additional color channels, one channel per fluorochrome. Thus, a sample containing both DAPI and Alexa Fluor 594 fluorochromes will require two channels.
   5. Use the "Image Properties" tool to set correct pixel (voxel) dimensions (XYZ), as determined in 3.7 above.
   6. Use "Display Adjustment" tool to change channel colors as needed (e.g., set DAPI channel to blue, Alexa Fluor 594 channel to red).
   7. To position the 3D volume in the image window, use the computer mouse to click and drag volume as required.
   8. Use the "Snapshot" tool in the toolbar to generate screenshots of the 3D image.
   9. Use the "Animation" tool in the toolbar to generate movies of 3D sample rotation.

Figure 1: Preparation of the Imaging Chamber. A: Consumables required for the preparation of the imaging chamber are blue tack, play dough or similar, and 2 coverslips (24 x 50 mm). B: Prepare two thin strips of blue tack (diameter approximately 2 mm x 2 cm), and two coverslips. C: Place blue tack strips on one coverslip, allowing enough space for the skin biopsy. Place the skin biopsy in a drop of CUBIC2 solution in between the two strips. D: Place the second coverslip on blue tack strips to cover the skin biopsy.