ab68 at a 1/1000 dilution staining Involucrin in human normal skin tissue by Immunohistochemistry (formalin fixed, paraffin embedded sections), incubated for 2 hours at 21°C. Heat mediated antigen retrieval step performed using citrate buffer pH 6.0. Blocked with 10% serum for 30 minutes at 21°C. Secondary used at 1/100 polyclonal Donkey anti-mouse IgG conjugated to Alexa Fluor 594 (red).

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KLC (keratinocyte-like cells) have the capacity to stratify

Human skin was decellularized and confirmed complete cell removal by H&E staining. ASC (human adipose-derived stem cells) were seeded on the epithelial side of human dermal decellularized matrices in the presence or absence of EGF (0.005 µg/mL). Human dermal decellularized matrices seeded with keratinocytes were used as controls. All three groups were kept in the same media conditions and lifted to an air-liquid interphase at the same time. Histological analysis revealed the presence of several layers of KLC with an organization similar to that of keratinocytes (A.1and 3). Surprisingly, ASC without EGF not only did not transdifferentiate into KLC, but instead migrated inside the matrix (A.2). Immunohistochemical analysis revealed a positive staining of cytokeratin-5 (B.3), cytokeratin-10 (C.3) and Involucrin (D.3) in stratified KLC matrices comparable to that of the stratified keratinocyte matrix (A, B, C and D.1). No positive staining was found in the ASC seeded matrices (A, B, C and D.2).

Overlay histogram showing A431 cells stained with ab68 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab68, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in A431 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab68 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab68, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Immunohistochemical analysis of Formalin fixed paraffin embedded human tonsil section labelling Involucrin with ab68 at dilution of 0.1 ug/mL.