Ab622557

Paraffin-embedded tissue sections were cut to 100 μm thick, dried, deparaffinized and rehydrated following standard protocols. The sections were incubated with primary antibodies against Beclin1 (1:500; ab622557; Abcam, Cambridge, MA, USA), LC3 (1:2,000; ab51520; Abcam) and LAMP2 (1:1,000; ab25631; Abcam) at 4°C overnight. Then, the sections were incubated with secondary antibodies for 30 min at room temperature. The sections were stained by diaminobenzidine (DAB; Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were lightly stained with hematoxylin. For negative control, the sections were treated as above but PBS (Hyclone, South Logan, UT, USA) instead of primary antibodies.
For each section, three fields were randomly selected (×200). The expression scores of Beclin1, LC3, and LAMP2 were on the grounds of staining intensity (no coloring: 0 point; light yellow: 1 point; brown yellow: 2 points; sepia: 3 points) and percentage of positive tumor cells (0–5%: 0 point; 6–25%: 1 point; 26–50%: 2 points; >50%: 3 points) (17 (link)). The final score was determined by staining intensity score × percentage of positive tumor cells (>4 scores: positive and 0–3 scores: negative).

Tissues were lysed using 300 μl RIPA plus 3 μl protease inhibitor PMSF (Beyotime, Beijing, China) on the ice. After centrifugation for 5 min at 12,000 × g, the supernatant was stored at −20°C. The protein concentration was determined using a BCA protein assay kit (Beyotime). Total proteins in the supernatant were subjected to separation in 10% SDS-PAGE, followed by transference onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk powder lasting 1 h at room temperature. Afterwards, the membrane was incubated by primary antibodies at 4°C overnight, and incubated with corresponding HRP-conjugated secondary antibodies (1:2,000; Abcam) for 50 min at room temperature in the dark. The primary antibodies included anti-Beclin1 (1:2,000; ab622557; Abcam), anti-LC3 (1:1,000; ab51520; Abcam), anti-LAMP2 (1:500; ab25631; Abcam) and anti-GAPDH (1:8,000; ab9485; Abcam). GAPDH was used as an internal control. The protein blot was visualized using Odyssey CLx.