Sc 56

Patient-derived explants (PDEs) from 5 ER+ primary breast tumors were processed and cultured as described in [50 (link)]. PDEs were treated with 100nM fulvestrant, 10μM riluzole, the combination, or solvent control (DMSO) for 48 hours before formalin fixation, paraffin embedding, sectioning, and staining for PCNA (1:1000, Santa Cruz Biotechnology Cat# sc-56, RRID:AB_628110), cleaved caspase 3 (1:300, Cell Signaling Technology Cat# 9661, RRID:AB_2341188), and Ki67 (1:500, Abcam Cat# ab16667, RRID:AB_302459). Stained sections were then visualized and scored as described in [50 (link)]. Data are presented as change relative to vehicle (set to 0) for each PDE.

Hematoxylin–eosin (HE), IHC, and immunofluorescence analyses were performed as previously described (Li et al., 2020 (link)). For IHC analysis, primary antibodies against cleaved Caspase-3 (Cell Signaling Technology, #9661), horseradish peroxidase (HRP)-conjugated secondary antibody (ZSbio, PV9001), and 3,3′-diaminobenzidine colorimetric reagent (ZSbio, ZLI-9018) were employed to detect the number of apoptotic cells. For immunofluorescence staining, primary antibodies against PCNA (1:200, Santa Cruz Biotechnology, sc-56) and Ki-67 (1:200, Cell Signaling Technology, #9129) and Alexa Fluor 555-labeled anti-rabbit (Beyotime, A0453), Alexa Fluor 555-labeled anti-mouse (Beyotime, A0460), and Alexa Fluor 488 anti-mouse IgG (ZSbio, ZF-0512) secondary antibodies were used to mark the proliferating cells. Slides were subsequently mounted with Vectorshield containing DAPI (H-1200, Vector Laboratories) and examined under a laser scanning confocal immunofluorescence microscope (LSM510 Exiter, Carl Zeiss).