Ab15481

Uterine sections from paraffin-embedded tissues were cut at 5 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series before blocking with 10% normal goat serum in PBS (pH 7.5) and incubating with primary antibody diluted in 10% normal goat serum in PBS (pH 7.5) overnight at 4°C at the following dilutions: 1:500 for anti-ARID1A (Sc-98441, SantaCruz), 1:100 for anti-Ki67 (ab15580, Abcam), anti-ESR1 (DAKO Corp.), 1:100 for anti-phospho-ESR1 (Ab31477, Abcam), 1:1000 for MUC-1 (ab15481, Abcam), 1:2000 for LTF (07–682, Millipore), MA), 1:20000 for MCM2 (Sc-9839, SantaCruz), 1:20000 for MCM6 (Sc-9843, SantaCruz), 1:5000 for KLF4 (Sc-20691, SantaCruz), 1:5000 for FLK15 (ab2647, Abcam), and 1:1000 for anti-total PGR antibody (A0098, DAKO Corp.). On the following day, sections were washed in PBS and incubated with the appropriate species-specific HRP-conjugated secondary antibody (2 μg/ml; Vector Laboratories) for 1 hr at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB kit (Vector Laboratories). A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities as previously described [101 (link)]. The number of PGR and Ki67-positive cells was counted in 200 epithelial cells and eight random fields of stromal cells.

Cell protein was collected by RIPA lysis buffer (KeyGEN, Nanjing, China) containing 1 nM PMSF (Biotool, Houston, TX, USA). 20 μg protein per well was electrophorsed in 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and incubated with different primary antibodies, including GALNT3 (16716–1-AP, Proteintech, China), caspase3 (ab13847, Abcam, the UK), cleaved caspase3 (ab13585, Abcam, the UK), PARP (ab74290, Abcam, the UK), cleaved PARP (ab4830, Abcam, the UK), MUC1 (ab15481, Abcam, the UK), PI3K-p110α (21890–1-AP, Proteintech, China), p-AKT 308 (AP3743a, Abgent, China), p-AKT 473 (AP3434a, Abgent, China), AKT (ab8805, Abcam, the UK), NF-κB (AP50006, Abgent, China) and GAPDH (AP7873a, Abgent, China), at 4 °C overnight. The membrane was treated with anti-rabbit IgG at 37 °C for 2 h. All bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by Lab Works (TM ver4.6, UVP, Bio Imaging Systems, NY, USA). GAPDH was used as control.

Histopathological analysis was performed by using hematoxylin and eosin (H&E) staining. Immunohistochemical analysis of HER2 was performed to evaluate cell proliferation and downstream intracellular signaling in neoplastic tissue. Primary antibodies used in this study were anti-HER2 (1:200, CST#4290, Cell Signaling Technology, Danvers, MA, USA), anti-MUC1 (1:100, ab15481, Abcam, Cambridge, MA, USA), anti-MUC2 (1:200, sc-15334, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUC5 (1:100, sc-21701, Santa Cruz Biotechnology), anti-Ki67 (1:100, ab16667, Abcam), anti-CyclinD1 (1:25, CST#2978, Cell Signaling Technology), anti-phospho-p44/p42 (1:100, CST#4376, Cell Signaling Technology), anti-SOX-9 (1:100, sc-20095, Santa Cruz Biotechnology), anti-TP53 (1:200, CST#2524, Cell Signaling Technology), and anti-CK19 (1:50, sc-376126, Santa Cruz Biotechnology).

Fixed salivary glands were first permeabilised in 0.5% Triton 100 × in PBS for 4 h and then blocked for 1 h in 2% donkey serum and 0.5% Triton-100X diluted in PBS (blocking solution). After blocking, salivary glands were incubated with the primary antibody diluted 1:100 in the blocking solution. E13.5 to E15.5 glands were incubated for 4 days shaking at 4 °C, while E16.5 to E18.5 were incubated for 7 days shaking at 4 °C. For the secondary antibody staining, salivary glands were first washed six times for 1 h in 0.5% Triton-100 × PBS at room temperature to remove the primary antibody. Then salivary glands were incubated with the secondary antibody diluted 1:500 in blocking solution for 5 days, shaking at 4 °C. Following the incubation, the tissue was washed six times for 1 h in 0.5% Triton-100 × PBS at room temperature and mounted on 22 × 32 mm coverslips with a 0.25 mm i-spacer (Sunjin Lab) in RapiClear 1.52 (SunJin Lab). The mounted tissue was incubated overnight at 4 °C to allow clearing of the samples and then imaged on the confocal microscope. The primary antibodies used were rabbit anti-laminin (L9393, Sigma), rabbit anti-Muc1 (ab15481, Abcam), anti-Mist1 (ab187978, Abcam) and β-catenin (L54E2) Alexa Fluor 488 Conjugate (#2849, Cell Signalling). The secondary antibody used was Alexa Fluor 647 Donkey Anti-Rabbit (A31573, Thermo Fisher Scientific).

Immunostaining was performed in 10% formalin-fixed paraffin-embedded sections (6 µm), using antibodies to Ki67 (Thermo Fisher Scientific, SP6), MUC1 (Abcam, ab15481), ERα (Abcam, ab810922), PGR (Abcam, ab63605), total STAT3 (Santa Cruz, sc-8019) and phosphorylated STAT3 (phospho Y705, Abcam, ab76315). As an isotype control, rabbit IgG (Dako, IS600) was used.