Materials and Reagents

MMI MembraneSlides^TM (MMI GmbH, catalog number: 50102)
MMI IsolationCaps transparent, 0.5 mL (MMI GmbH, catalog number: 50204)
Phospholipase A2 (Sigma-Aldrich, catalog number: P6534)
Sodium deoxycholate (Sigma-Aldrich, catalog number: V900388)
PBS (TBD Science, catalog number: PB2004Y)
EDTA (Sigma-Aldrich, catalog number: E9884)
NaCl (Sigma-Aldrich, catalog number: S9888)
DNase (Sigma-Aldrich, catalog number: 11284932001)
RNase (Sigma-Aldrich, catalog number: 10109134001)
OCT compound (SAKURA, catalog number: 4583)
4% paraformaldehyde (BOSTER Biological Technology, catalog number: AR1068)
Sucrose (Sigma-Aldrich, catalog number: V900116)
Delipidation solution (see Recipes)
DNase-RNase solution (see Recipes)
3.4 M NaCl solution (see Recipes)
30% sucrose solution (see Recipes)

Equipment

Laser microdissection system (MMI, cellcut plus)
Freezing microtome (Leica, model: CM1950)
Shaker (tcsysb, THZ-C)
Scalpel (BELEVOR MEDICAL, catalog number: 03.0030.01)

Procedure

Obtain native frozen skin tissue sections
1. Rinse skin tissue three times with cold PBS.
2. Fix tissue with 4% paraformaldehyde at room temperature for 24 h.
3. Perform dehydration with 10 mL of 30% sucrose solution at 4 °C until the tissue sinks to the bottom.
4. Freeze at -80 °C and then embed the skin tissues. Apply a layer of OCT compound in the groove of the embedding mold and place the tissue section down into the groove. Fill with embedding agent (OCT compound) and put the sample on the freezer until the embedding agent solidifies.
5. Cut 20-μm-thick sections of native skin tissues. Place the MembraneSlides^TM (with the MMI logo face up) close to the slice, which will stick to it. Reverse the MembraneSlides^TM, gently press them at the corresponding position below the slice with your fingers, and the sample will completely adhere to the MembraneSlides^TM.
6. Mount sections on MMI MembraneSlides^TMand store at -20 °C.

Obtain decellularized frozen skin tissue sections
1. Rinse skin tissue three times with cold PBS containing 0.1% EDTA.
2. Perform delipidation by treating tissue with 25 mL of delipidation solution (see Recipes) for 4 h in a shaker at 37 °C until the tissue segments become oyster white.
3. Gently scrap the surface of the skin with the back of the scalpel to remove the epidermis.
4. Rinse the decellularized dermal scaffolds with 3.4 M NaCl (see Recipes) at 37 °C for 1 h.
5. Wash the samples with DNase-RNase solution (see Recipes) at 37 °C for 1 h.
6. Rinse skin tissue three times with cold PBS.
7. Fix tissue with 4% paraformaldehyde at room temperature for 24 h.
8. Perform dehydration with 10 mL of 30% sucrose solution in a 15 mL centrifuge tube at 4 °C until the tissue sinks to the bottom.
9. Freeze and embed the skin tissues as in step A2.
10. Cut 20-μm-thick sections of decellularized skin tissues as in step A3.
11. Mount sections on MMI MembraneSlides^TMand store slides at -20 °C.

Laser capture microdissection (LCM) of frozen skin sections
1. Open LCM system. Install MembraneSlides and IsolationCaps transparent.
2. Switch to 4× objective and start slide scan and navigation.
3. Switch to 10× objective for subsequent dissection.
4. Set laser position indicator. Click the button to fire the laser and check whether the laser position matches the green cross. If not, click “CellCut” – “Laser” – “Set Laser Position” and click at the location where the laser is emitted.
5. Calibrate the parameters for the laser. Select the freehand tool, draw a long line on the empty area without tissues on the slides, and cut. Adjust “Cut velocity,” “Laser focus,” and “Laser power” to make the cutting line clear and precise.
6. Move to the target position, draw cut lines, and start cutting. Increase the cut velocity, cut power, and number of cuts appropriately to get the best cutting efficiency.
7. Pick up the sample under manual mode. Click the collecting button to attach the cutout to the EP tube cover and check if the tissue was successfully collected. If not, repeat collection or try to adjust the position of the EP tube and even recut.
8. Repeat steps C6 and C7 to isolate stratum corneum, granulosum-spinosum, basal layer, superficial dermis, and deep dermis on native sections and basement membrane on decellularized sections successively.
9. Check the entire slice to ensure that the target area is cut and collected.
10. Label the samples and store them at -20 °C for subsequent MS analysis.

Notes

To obtain a clearer view of tissue structure when cutting, hematoxylin-eosin staining can be performed on the sections.
The LCM systems allow simultaneous cutting of the same structure for up to 3–4 slices and collection of multiple structures with multiple tubes by using the group function.
When the sample is difficult to be dissected or collected by the EP tube cover, first check if the objective magnification is correct. Then, optimize laser parameters, such as increasing laser power or slowing down the cutting speed, and repeat the cuts. Finally, move the position of the EP tube cover to an area with less adherent samples or replace the collection tube with a new one.
The cutting circle should not be too small, or the tissue will be easily repelled away by the laser. Also, the cutting line should not be too long, or the tissue will break during collection.
For laser parameters calibration, first decrease “Cut velocity” and “Laser power” to a low value (as 5 μm/s and 5%) and cut. Then, change “Laser focus” until the cutting line becomes clear and spacious. Finally, decrease the “Laser power” to get a fine line and change the “Laser focus” again to find the finest line. After calibration, increase the “Cut velocity” to a high value (as 70 μm/s) and increase the “Laser power” accordingly to get a fast and precise cutting line. For best cutting efficiency, change “Cut velocity” and “Laser power” according to different tissue locations and thickness.
Videos 1–5 show the continuous process of cutting the stratum corneum, stratum granulosum-spinosum, basal layer, superficial dermis, and deep dermis of the native skin tissue sections from outside to inside. Video 6 shows the process of cutting the basement membrane of the decellularized skin tissue sections.

Video 1. Isolation of stratum corneum

Video 2. Isolation of stratum granulosum-spinosum

Video 3. Isolation of basal layer

Video 4. Isolation of superficial dermis

Video 5. Isolation of deep dermis

Video 6. Isolation of basement membrane

Recipes

Delipidation solution
1. Dissolve 10 g of sodium deoxycholate powder in 1 L of deionized water.
2. Add phospholipase A2 to above solution before delipidation treatment to make a final concentration of 2,000 U/L.

DNase-RNase solution
Dissolve 1 mg of DNase powder and 0.5 mg of RNase powder in 100 mL of PBS buffer before use.

3.4 M NaCl solution
1. Dissolve 198.9 g of NaCl powder with 800 mL of deionized water.
2. After dissolving, fill with deionized water to 1 L.

30% sucrose solution
1. Dissolve 300 g of NaCl powder with 800 mL of deionized water.
2. After dissolving, fill with deionized water to 1 L.

Acknowledgments

This work was supported by Beijing Municipal Science and Technology Commission (Z191100006619011), Capital’s Funds for Health Improvement and Research (2020-2-4016), CAMS Innovation Fund for Medical Sciences (CIFMS 2020-I2M-C&T-B-048), and National Science and Technology Major Project (2021YFA1301603).

This protocol was adapted from Li et al. (2022).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics

The authors declare no conflict with respect to ethical grounds.

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