*4.3. Histological Analyses*

Biopsies of skin substitutes were analyzed by histological and immunohistochemical methods after 21 days of culture at the air-liquid interface. For living epidermal thickness analyses, two biopsies of each condition were fixed in Histochoice® (AMRESCO, Inc., Solon, OH, USA) and embedded in paraffin wax. After that, deparaffinized 5 μm tissue sections were cut and stained with Masson's Trichrome. The thickness of the living epidermis was obtained by measures made with Image J software (National Institutes of Health, USA, http://imagej.nih.gov/ij). Ten measurements in three different parts of each skin substitute were used to compare the living epidermis thickness between skin substitutes.

#### *4.4. Indirect Immunofluorescence on Frozen Tissues*

For immunofluorescence staining, tissues were embedded in Tissue-Tek O.C.T Compound (Sakura Finetek, CA, USA) and 6 μm thick sections were fixed in acetone at −20 ◦C before staining. The following antibodies were used and incubated in a dark room at room temperature for 45 min: rabbit anti-involucrin (Abcam, Cambridge, MA, USA), rabbit anti-filaggrin (Abcam, Cambridge, MA, USA), rabbit anti-keratin 10 (Abcam, Cambridge, MA, USA), mouse anti-Ki67 IgG1 (BD Biosciences, CA, USA), rabbit anti-AC9 (ab191423, Abcam, Cambridge, MA, USA) and rabbit anti-beta 2 adrenergic receptor (ab61778, Abcam, Cambridge, MA, USA). Tissues were then incubated with Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 goa<sup>t</sup> anti-mouse IgG (1:1600, Thermofisher Scientific, CA, USA) for 30 min also at room temperature. Nuclear counter staining using DAPI (SouthernBiotech, AL, USA) was then effected on different samples. Each tissue was observed using a Zeiss Axio Imager M2 microscope with an AxioCam ICc1 camera. The quantification of immunofluorescence staining was performed by densitometry using ImageJ software (from Wayne Rasband, National Institute of Health (NIH), USA).

#### *4.5. Cyclic AMP Competitive ELISA Kit on Frozen Tissues*

After 63 days of cell culture to prepare the reconstructed skin substitutes, the epidermis was mechanically separated from the dermis using scalpel and forceps, and samples were quick-frozen in liquid nitrogen to preserve the tissue integrity. Tissues were crushed in a Safe-Lock 2.0 mL Eppendorf tube (ATS Scientific, Inc., Burlington, ON, Canada) with two 6 mm stainless ball using a Cryomill MM400 (Retsch®, Newtown, PA, USA). The levels of cAMP were assayed using a Cyclic AMP Competitive ELISA Kit (ThermoFisher Scientific, Vienna, Austria). The non-acetylated version of the ELISA Kit was used following the protocol provided by the manufacturer, and 400 μg of cell lysate was used in the test. Total protein concentrations were determined using a BCA Protein Assay Kit according to the manufacturer's instructions (Thermo Scientific, Rockford, IL, USA).
