Ab3576

Cells were seeded in 6-well plates and maintained for 48 h. After being washed 3 times with PBS, cells were fixed in 4% paraformaldehyde solution for 30 min, permeabilized with 0.3% Triton X-100 for 10 min, and suspended in 10% normal goat serum (Gibco Life Technologies, USA) for 10 min, then incubated with the primary antibodies: α-Actin (1:1,000, Abcam Cat# ab69512, RRID:AB_2222725), ER-α (1:1,000, Abcam Cat# ab3743, RRID:AB_304042), and ER-β (1:1,000, Abcam Cat# ab3576, RRID:AB_303922) overnight at 4 °C. After another three washes with PBS, the cells were incubated with secondary antibody for 1 h at room temperature. DAPI (4',6-diamidino-2-phenylindole) reagent (Shanghai Leuven Biotechnology Co., Ltd., China) was used to counterstain the nuclei. The result of the staining was observed with an inverted fluorescence microscope system (OLYMPUS, X53, Olympus Corporation, Japan).

We obtained paraffin-embedded ccRCC specimens from the Department of Pathology for 93 ccRCC cases from The Affiliated Hospital of Qingdao University. IHC staining was carried out as described previously [30 ]. Briefly, fresh tissues were fixed in formalin and embedded in paraffin, then sectioned at a thickness of 5 μm. The sections were dewaxed in xylene and rinsed in alcohol and in graded alcohol/water mixtures. Hydrogen peroxide (3%) was applied to block the endogenous peroxidase activity. The sections were subsequently treated in a microwave oven twice for 6 min in citrate buffer (pH 6.0) at 600 W to undergo antigen repairing. After blocking with goat serum for 30 min, sections were incubated with primary antibodies against estrogen receptor-β (ER-β) (ab3576) (1:50) and IGF-1R (ab39398) (1:100) (Abcam, Cambridge, MA, USA) at 4 °C overnight, followed by anti-mouse/rabbit horseradish peroxidase-labeled antibody (Univ-bio, Shanghai, China) as the second antibody. ER-β and IGF-1R staining were scored as 0, 1, 2, and 3 according to the proportion of positively stained cells and the intensity of the staining, as described previously [23 (link)].

Estradiol (E2758, purity ≥ 98%), MPP dihydrochloride hydrate (M7068, purity ≥ 97%), PHTPP (SML1355, purity ≥ 98%) and 3-Methyladenine (3-MA, M9281, purity ≥ 99%) were purchased from Sigma (Saint Louis, USA). Antibody for NLRP3 (19771-1-AP) and IL-18 (10663-1-AP) were purchased from Proteintech (Wuhan, China). Antibody for caspase-1 (48847) was purchased from SAB signalway antibody (Maryland, USA). Antibody for GSDMD (A18281) and IL-1β (A16288) were purchased from ABclonal (Wuhan, China). Antibody for Beclin 1 (ab210498), LC3B (ab192890), SQSTM1 (ab56416), Hcy (ab15154), estrogen receptor alpha (ERα) (ab32063), estrogen receptor beta (ERβ) (ab3576), GSDMD (ab219800), and β-actin (ab8226) were purchased from Abcam (Cambridge, UK). FITC - goat Anti-mouse IgG (ab6785), TRITC - goat anti-rabbit IgG (BS10250), FITC - goat anti-rabbit IgG (BS10950), HRP - goat anti-rabbit IgG (BS13278), and HRP - goat anti-mouse IgG (BS12478) were purchased from Bioworld (Bloomington, USA). The prestained protein MW marker (26617) were purchased from Thermo scientific. Control siRNA and ERα siRNA (sc-29305) were purchased from Santa Cruz (Santa Cruz, USA).

The sections were heated in a microwave oven in citrate buffer (pH =6.0) for 20 minutes at 95°C and cooled at room temperature. The sections were then washed with phosphate buffered saline (PBS) for three times (3 minutes/time). Endogenous peroxidase activity was blocked in 3% H₂O₂ for 20 minutes at room temperature. After washing with PBS for three times (3 minutes/time), sections were incubated with normal goat serum for 30 minutes at room temperature. The sections were then incubated overnight at 4°C with specificity primary antibody (anti-ERα [ab37438], Abcam, UK; anti-ERβ [ab3576], Abcam; PI3 kinase p110α [C73F8], Cell Signaling, USA; AKT [C73H10], Cell Signaling; E-cadherin [24E10], Cell Signaling; vimentin [D21H3], Cell Signaling) 37°C rewarming for 45 minutes. The sections were washed three times with PBS (3 minutes/time), then incubated with GTVision (Gene Technology Shanghai Co., Ltd., Shanghai, People’s Republic of China) for 30 minutes at 37°C. The sections were then washed three times with PBS and visualized with diaminnobenzidinetetra-hydrochloride (DAB kit; Shanghai Secco Chemical Technology Co., Ltd., People’s Republic of China). Finally, the sections were counterstained with hematoxylin and dehydrated.