Sc 18835

Western blotting was performed as previously described [2 (link)] using primary antibodies raised against P-glycoprotein (1:100, sc-13131: Santa Cruz Biotech, CA, USA), MRP-1 (1:200, sc-18835, Santa Cruz Biotech), MDM2 (1:100, IF2; EMD Chemicals Inc., Gibbstown, NJ, USA), p53 (1:300, DO-7; Novocastra Laboratories, Newcastle, UK), serine 15 pp53 (1:1000, # 9284; Cell Signalling, Danvers, MA, USA) and p21^WAF1 (1:100, EA10, EMD Chemicals Inc). α-Tubulin (DM1A; Sigma-Aldrich, Dorset, UK) was used as a marker of protein loading.

MRP1-positive cells were measured by flow cytometry (FACS Jazz; BD Biosciences, Franklin Lakes, NJ, USA) using the protocol previously described by Torres et al. [3 (link)]. Briefly, non-GSCs and GSCs were treated with vehicle (1× PBS) and FK506 (7, 15, and 30 ng/mL) (Cat. No. 3631; Tocris Bioscience, Bristol, UK) for 24 h. Cells were fixed with PFA (paraformaldehyde) 3.7%, blocked (1× PBS-BSA (bovine serum albumin) 0.5% at room temperature) and marked with anti-MRP1 (sc-18835, Santa Cruz Biotechnology) antibody followed by a secondary anti-mouse Alexa 488 (Life Technologies). Lastly, events were acquired through the FL1 filter of the cytometer.

Histological sections were immunohistochemically stained to visualise the MRP1 expression and to study the proliferative state of the tumours. Microtome sectioned tissue specimens of 5 µm were dehydrated at 50 °C overnight, followed by deparaffinisation and rehydration. Rehydrated slides were first incubated in sodium citrate buffer pH 6 at 95 °C for 30 min for antigen retrieval and cooled down to room temperature. For ki67 staining, slides were additionally incubated with 0.1% Triton X-100 in PBS for 10 min to permeablise the nuclear envelope. Slides were then incubated in 1% H₂O₂ for 15 min to quench endogenous peroxidase activity, followed by serum blocking for 1 h. Goat anti-mouse IgG Fab (Abcam, ab6668) was added for 1 h to block endogenous IgG. After washing in PBST, slides were incubated with primary against ki67 (Abcam, ab8191) or primary against MRP1 (Santa Cruz, sc-18835) overnight in a moisture chamber at 4 °C. Slides were then incubated with biotinylated mouse-rabbit polyvalent secondary (Abcam, ab64264), followed by streptavidin peroxidase (Abcam, ab64264) incubation. After washing, slides were developed using 3,3′-diaminobenzidine (DAB) substrate for 15 min then counterstained with Mayer’s haematoxylin QS (Vector, H-3404), and eventually mounted in coverslips with DPX.