Nuclear fast red

HUVEC were passaged once after cryorecovery, cultured for 24 h, treated with indicated doses of palbociclib or abemaciclib in water vehicle for 72 h, and then collected for downstream processing. A separate aliquot of untreated HUVEC in exponential growth phase was collected alongside for reference comparisons. Cell cycle state was assessed for three independent replicates of one million cells per treatment condition, quantified using the Vendelov method [26 (link)]. Staining for senescence-associated β-galactosidase (SA-β-gal) was carried out on three independent replicates per treatment condition using the Cell Signaling Technologies SA-β-gal staining kit according to the manufacturer’s instructions. After developing the stain for 48 h, the cells were fixed using 4% paraformaldehyde in PBS and counterstained with Nuclear Fast Red (Thermo Scientific, Waltham, MA, USA). Images were acquired using an Olympus BX40 microscope. Positive and negative cells per field were quantified in a blinded manner using QuPath v0.3.2 [24 (link)].

Histological analysis of miR-21 was performed via in situ hybridization with a miRCURY LNA™ microRNA ISH Optimization Kit (Exiqon, Vedbæk, Denmark) as per the manufacturer's instructions. Briefly, sample slides were deparaffinized and stained with miR-21 probes. Cell nuclei were counterstained with Nuclear Fast Red™ (Thermo Fisher Scientific, Waltham, MA, USA). Expression of miR-21 was ranked based on H score ranging from 0 to 3, formulated as ((percentage of 0) × 0) + ((percentage of 1+) × 1) + ((percentage of 2+) × 2) + ((percentage of 3+) × 3) (0, nil; 1+, low or weak; 2+, moderate; and 3+, high or strong) [31 (link)]. For histological determination of ASPP2 and cleaved caspase 3 expression, IHC was performed with a Novocastra™ Manual Detection System kit (Leica Biosystems, Newcastle, UK) as described previously [31 (link)]. High expression was defined as positive staining in ≥50% of the cells.

Sim1 expression was examined by in situ hybridization using a previously described probe (Fan et al. 1996 (link)). The probe was labeled with digoxigenin (DIG) using DIG RNA labeling mix (Roche 11277073910) following the manufacturer's instructions. The in situ hybridization procedure followed that outlined in Prado et al. (2004) (link) with the following modifications: Ten-micrometer sections were postfixed for 1 h at room temperature, and Proteinase K treatment was omitted. Bound probes were visualized with BM Purple AP substrate (Roche 11442074001) and counterstained with Nuclear Fast Red (Thermo Fisher Scientific NC9483816).