Materials and Reagents

24-well cell-culture treated plate (ThermoFisher, catalog number: 142475)
Ethanol 70%
5 × 5 sterile gauze pads
8 × 8 mm Peel away embedding mold (Electron Microscopy Sciences, catalog number: 70180)
Superfrost Plus microscope slides (75 × 25 mm, Fisher Scientific, catalog number: 12-550-15)
Microscope cover glass (24 × 60 mm, Marienfeld, catalog number: 0101242)
Dulbecco′s Modified Eagle′s Medium, high glucose (DMEM) (Sigma-Aldrich, catalog number: D5796)
Dulbecco's-Phosphate-buffered saline (DPBS) (pH 6.7–7) (ThermoFisher, catalog number: 14200075)
Incubator 37°C, CO₂ 5% (ThermoFisher, Heracell 150i, catalog number: 50116048)
OCT compound (Scigen, catalog number: 4586)
Formaldehyde 35% (Bio-Lab, catalog number: 67505)
ImmEdge Hydrophobic Barrier PAP Pen (Vector Laboratories, catalog number: H-4000)
DAPI (Sigma-Aldrich, catalog number: D9542)
Fluoromount mounting medium (Sigma-Aldrich, catalog number: F4680)

Equipment

Dissecting scissors, 9.5 cm, straight (WPI, catalog number: 503244)
Dressing forceps, 12.5 cm, serrated (WPI, catalog number: 501217)
Tweezers
Small beaker
Cryostat (Leica CM1950)
Glass staining jar (Normax, supplied by Alexred, catalog number: NR 4431000)

Software

ImageJ freeware (http://rsbweb.nih.gov/ij/) – image analysis

Procedure

Skin sample preparation
1. Harvest skin and maintain it in DMEM with 1% Penicillin/Streptomycin at 4°C until use.
2. For storage, use a plate with a cover in a suitable size for your skin sample. Adjust the medium volume to moisturize the dermis. Do not cover the external skin layers (epidermis) with the medium (see the example in Figure 1a).
3. Use the full-thickness human skin within 24 h of harvesting.
4. Remove fats with dissecting scissors and forceps gently. Do not leave any fat remains, and do not harm the dermis.
5. Remove hair's edges with scissors.
6. Cut ~2.5 cm² square skin samples.
7. Sterilize the skin samples in a small beaker with 70% ethanol solution for 1 min.
8. After sterilization, place the samples in PBS 1× for 10 s, to neutralize the ethanol.
Treatment with nanoparticles
1. Take a 24-well plate and fill a well with DMEM, up to the well walls.
2. Select a skin sample with dense hair follicles and place it on the well edge, with the dermis facing the medium. The skin sample should surround the well edges without falling into it (Figure 1).
3. Apply the desired fluorescent nanoparticles in the required form (solution, ointment, gel, etc.) on the skin samples.
4. Cover the plate and incubate the tissue in an incubator at 37°C for 24 h.
5. Take the skin samples from the plate and remove excess particles ointment/solution with a sterile gauze pad soaked with PBS.
Figure 1. Skin storage and experimental setup.
A. Skin samples storage before use within 24 h from harvesting. Skin sample should be placed in a vessel at a suitable size with DMEM in such volume that it does not cover the epidermis. B. Skin samples, cleaned from fats, are placed on 24 well plate edges for treatment. The well is filled with DMEM, to nourish the sample during incubation.
Taken from previous work with the permission of the publisher (Nanomedicine: Nanotechnology, Biology and Medicine. License number 5195761380089).
Cryo-sectioning—sample preparation and sectioning
1. Freeze the skin samples in OCT, using dry ice in an embedding mold. We recommend using one mold per sample.
2. For horizontal sectioning, place the sample with the epidermis towards the bottom, and cover it with OCT.
3. Set the chamber temperature to -21°C, and the specimen head temperature to -29°C.
4. Fix the sample on the disc.
5. Prepare a set of positive charged slides, and number each slide. Each slide will contain 2–3 sections. The numbers should be consecutive and represent the depth of the skin sample.
6. Cut the first 100 μm from the skin, to achieve a flat section that can be applied on microscope slides.
7. Cut sections at the thickness of 14 μm, according to the slides prepared in step C5. Repeat this step to the desired depth.
Sample staining for confocal microscopy
1. Fix slides in a glass staining jar with 4% formaldehyde for 20 min.
2. Wash three times with PBS 1× for 5 min, by dipping in a glass staining jar.
3. Use a PAP pen to mark a square around the sample.
4. Stain samples with 100 μL of DAPI (0.1 μg/mL) for 10 min.
5. Mount slides with one drop of fluoromount media per sample and cover with a cover glass.
6. Image samples with confocal microscopy (Figure 2).
7. Analyze images using ImageJ software.
8. Split image channels for each skin layer (Image>Color>Split Channels).
9. To measure fluorescence intensity and reduce background for further analysis (Figure 2c, 2d), choose the required area with the oval selector and use the measurement tool (Analyze>Measure).
10. Repeat step D9 with the oval selector on empty area, for background intensity measurement. In your data analysis, reduce the background intensity from your measurements.
  
  Figure 2. Skin samples horizontal microscopy.
  A. Illustration of horizontal skin segmenting B. Two consecutive skin samples in the horizontal orientation under confocal fluorescent imaging. Blue DAPI staining reveals hair follicles as circles in the center. Fluorescent nanoparticles accumulate around the hair (purple). C. Image analysis by ImageJ is used to split channels and calculate fluorescent intensity to quantify particle accumulation per section. (D). Taken from previous work with the permission of the publisher (Nanomedicine: Nanotechnology, Biology and Medicine. License number 5195761380089).

Acknowledgments

This project was funded by a Kamin fund of the Israel Innovation Authority, and by an Israel Science Foundation (ISF) grant. This protocol served as in our previous work "Physical properties of gold nanoparticles affect skin penetration via hair follicles" (Friedman et al., 2021).

Competing interests

The authors declare no competing interests.

Ethics

Human full-thickness skin samples were received from patients undergoing routine plastic surgery procedures in the plastic surgery unit at "Hadassah Medical Center" after receiving written consent, with approval by the Hadassah Research Ethics Committee, in adherence to Helsinki guidelines (0348-17-HMO).

References

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Friedman, N., Dagan, A., Elia, J., Merims, S. and Benny, O. (2021). Physical properties of gold nanoparticles affect skin penetration via hair follicles. Nanomedicine 36: 102414.
Lohan, S. B., Saeidpour, S., Solik, A., Schanzer, S., Richter, H., Dong, P., Darvin, M. E., Bodmeier, R., Patzelt, A., Zoubari, G., et al. (2017). Investigation of the cutaneous penetration behavior of dexamethasone loaded to nano-sized lipid particles by EPR spectroscopy, and confocal Raman and laser scanning microscopy. Eur J Pharm Biopharm 116: 102-110.
Patzelt, A. and Lademann, J. (2013). Drug delivery to hair follicles. Expert Opin Drug Deliv 10(6): 787-797.