Pk 4000

Tissues were fixed in 10% (v/v) formaldehyde in PBS and embedded in paraffin. 5 μm sections were made and treated with boiling citrate buffer (pH 6.0) for 30 mins to complete antigen retrieval. After incubation with 3% peroxidase and 10% goat serum blocking buffer, the slides were incubated with 1:100 diluted anti-FOXH1 (ab189960, Abcam) or anti-p-S6K1 (9234, CST) primary antibody at 4° C for at least 16 hours. Then the slides were incubated with biotin-labeled secondary antibody for another 30 min, and followed by 30 min streptavidin incubation (PK-4000, Vectastain, USA.) The FOXH1 signal was determined by DAB staining.

Antigen retrieval was performed on rehydrated tissue sections in boiling citrate buffer (pH 6.0) for 30 min. After being treated with 3% H₂O₂ and blocking buffer (5% normal goat serum in PBS), tissue sections were incubated with p-STAT3 (Y705) (9131, Cell signaling), Ki67 (BD pharmingen) or CD133 (Proteintech) at 4 °C overnight. Then biotin-labeled secondary antibody was added for another 30 min, followed by 30 min incubation of streptavidin-horseradish peroxidase (HRP) (PK-4000, Vectastain, Burlingame, CA) and signals were visualized by peroxidase substrate DAB kit (SK-4100, Vectastain, Burlingame, CA).

The skin was placed in a 30% sucrose solution for cryoprotection. After 3 days in sucrose, the skin was frozen in liquid nitrogen. The tissue was sliced into sections and mounted on slides for staining. Slides were incubated at 4°C overnight with primary antibodies to AQP5 (1 : 1000), UT-A1 (1 : 1500), and UT-B1 (1 : 2500). Visualization was achieved using the standard ABC kit (Vectastain PK-4000) according to the manufacturer's instructions. Preincubation control experiments have been addressed to confirm that the antibodies used really are detecting the proteins of interest.

Western blotting, immunostainings and β-galactosidase were performed as previously described[8 (link)]. Antibodies for indicated antigens include beta-catenin 1:200 in PBS (AB6302, Abcam), insulin 1:800 in PBS (sc-9168, Santa cruz), glucagon 1:1200 in PBS (PA039-5P, BioGenex), e-cadherin 1:500 in MOM diluent (610182, BD Bioscience), beta-galactosidase 1:1000 in PBS (8559762, MP Biomedical), and amylase 1:5000 in Tris Buffered saline (Sc-46657, Santa Cruz). Reagents: DAPI (5mg/mL) 1:4000 in PBS (D1306, Thermofisher) and 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside 1mg/mL in PBS (Sigma-Aldrich, B4252-100MG) and mouse on mouse (MOM) basic kit (BMK-2202, Vector Laboratories) according to manufacturer’s descriptions, ABC basic kit 1:25 (PK-4000, Vector Laboratories) and 3–3’-Diaminobenzene (DAB) (SK-4100, Vector Laboratories) in the presence of 0.0005% (v/v) hydrogen peroxide (H₂O₂).

Fixed samples were embedded in paraffin and sectioned in a routine manner. The sections were subjected to hematoxylin-eosin (HE), Ki-67 and terminal dUTP nick-end labeling (TUNEL) staining.
For Ki-67 staining, tissue sections (3 μm) on glass slides were deparaffinized, washed with ethanol and water, and soaked in phosphate-buffered saline (PBS). The sections were autoclaved with 0.01 M citrate buffer (pH 6.0) for 15 min (121°C). The sections were then washed with PBS and incubated with rabbit polyclonal anti-Ki-67 antibody (1:50; E0468, Dako, Glostrup, Denmark) for 30 min at room temperature. After washing with PBS, the sections were incubated with rat anti-IgG antibody (1:100; sc-372; Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at room temperature. The slides were washed with PBS and stained using the ABC method (PK-4000; Vector Laboratories, Inc.) for 30 min.
For TUNEL staining, tissue sections (3 μm) on glass slides were deparaffinized, washed with ethanol and water, and soaked in diluted water. TUNEL staining was performed using the In situ Apoptosis Detection kit (Takara Bio, Inc., Shiga, Japan) according to the manufacturer’s instructions. Ten high-power fields were randomly selected and the positive cells were counted.

Free-floating immunohistochemistry (IHC) was performed using the streptavidin-biotin-peroxidase method as previously described [49 (link),78 (link)]. In brief, sections were quenched in a solution containing 3% hydrogen peroxide and 10% methanol in distilled water to eliminate endogenous peroxidase activity. Nonspecific antibody binding was blocked by incubation in a solution containing 3% normal goat serum in Tris-buffered saline (TBS) with 0.2% Triton X-100 at room temperature for 1 h. The primary antibody (mouse antihuman-α-synuclein, 1:10,000, 36-008, Merck Millipore, Cork, Ireland) was diluted in 1% serum in TBS with 0.2% Triton X-100 and allowed to incubate with the sections overnight. Sections were then incubated with the biotinylated secondary antibody (goat antirabbit, 1:200, 111-065-144, Jackson ImmunoResearch, Cambridgeshire, UK) with 1% serum for 3 h. A streptavidin-biotin-horseradish peroxidase solution (Vector PK-4000) was subsequently added to sections and allowed to incubate for 2 h. Development of the staining was performed using a 0.5% diaminobenzidine tetrahydrochloride (DAB) (Sigma D5637) solution in TBS containing 0.3 µL/mL of hydrogen peroxide. Sections were mounted onto gelatine-coated slides, dehydrated in an ascending series of alcohols, cleared in xylene and coverslipped using DPX mountant.

Sections were washed three times in PBS for 5 min, quenched in 0.3% H₂O₂ for 30 min, washed in 0.05% Triton X-100 in 1× PBS (PBS-T), then blocked in 3% normal horse serum and 2% BSA (Sigma-Aldrich) for 60 min at room temperature, followed by incubation in primary antibody at 4 °C, except for GDNF which was at room temperature (Supplementary Table 3). Sections were then washed with 0.2% PBS-T and placed in appropriate biotinylated secondary antibodies (Vector, BA-9500 or BA-1100, 1:200) for 2 h at room temperature. After washing, sections were incubated in avidin–biotin complex (Vector, PK-4000) for 45 min at room temperature and washed for 30 min in 1× PBS. A DAB kit (Vector, SK-4100) was used for chromogenic detection. For GDNF staining, samples were enhanced with nickel substrate coincubation. Sections were then washed in tap water and 1× PBS, mounted on slides and dehydrated in two washes each of 95% ethanol (EtOH), 100% EtOH and xylene. Images taken with Leica DM 2000 LED microscope using Leica ICC50 HD camera.