Immunofluorescence Staining in Serial Sections for K77 and K7/K14 in Eccrine Sweat Glands

Expression of S100A2 and S100P in human eccrine sweat glands and their application in differentiating secretory coil-like from duct-like structures in the

3D reconstituted eccrine sweat spheroids

Supplementary Material

Methods

Immunofluorescence staining in serial sections for K77 and K7/K14 in eccrine sweat glands.

Immunofluorescence staining was performed as follows. First, 5m-thinkness frozen sections were fixed in cold acetone at 4°C for 10 min and permeabilized in 0.1% Triton

X-100 for 10 min. Sections were incubated with goat serum in PBS for 30 min at

37°C to block nonspecific sites.

For sections staining for K77, the sections were incubated with rabbit anti-K77 (1:100 dilution, SC368918, Santa Cruz, USA) at 4°C overnight, followed by incubation with

Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibodies (1:500 dilution, A0516,

Beyotime, Shanghai, China) in the dark for 1 h.

For sections staining for K7/K14, the sections were incubated with rabbit anti-K14

(ZA-0540, ZSGB-Bio, Beijing, China) and mouse anti-K7(ZM-0071, ZSGB-Bio,

Beijing, China) primary antibodies together, and then with the secondary antibodies,

Alexa Fluor 488-labeled goat anti-mouse IgG (1:500 dilution, A0428, Beyotime,

Shanghai, China) and Cy3-labeled goat anti-rabbit IgG, together.

Finally, all the sections were counterstained with 5 μg/ml 4', 6-diamidino-2- phenylindole (DAPI, C1005, Beyotime, Shanghai, China) for 10 min at room temperature in the dark and mounted with antifade mounting medium (P0128,

Beyotime, Shanghai, China). PBS was used for rinsing between steps. Sections omitting the primary antibodies were used as negative controls. All sections were observed under an Olympus BX51 fluorescence microscope (Tokyo, Japan).

Results

According to the localization of K7 and K14 in human eccrine sweat glands in serial sections, K77 is indeed expressed in ducts, but it is also expressed in secretory coils of eccrine sweat glands. Secretory coils (arrowheads); duct (arrows) Fig S1. Immunofluorescence staining in serial sections for K77 and K7/K14 in eccrine sweat glands.

Referred to the localization of K7 and K14 in human eccrine sweat glands and skins, the staining showed that K77 is indeed expressed in luminal duct cells, but it is also expressed in secretory cells of eccrine sweat glands and epidermis. Secretory coils

(arrowheads); duct (arrows)