Anti anln

Formalin-fixed paraffin-embedded BLCA samples were cut into 5-μm-thick sections. Antigen retrieval and immunostaining of sections were performed as described previously^{37 (link)}. Primary antibody (1:100) anti-ANLN was purchased from Abcam (ab154337). Staining intensity was classified in a blinded fashion by pathologists. The immunohistochemical stain was scored on the percentage of positively tumor cell nucleus (negative, score 0; <1/3, score 1; 1/3–2/3, score 2; >2/3, score 3) and the color intensity of cytoplasm (negative, score 0; stramineous, score 1; buffy, score 2; dark brown, score 3). The two scores were combined, scores of 0–3 were defined as low expression, and 4–6 were defined as high expression. Immunofluorescence was performed using a standard protocol as described previously^{23 (link)}. Primary antibodies were as follows: anti-ANLN (1:100), anti-F-actin (1:100, Abcam, ab130935). Images of immunofluorescence were taken by confocal microscope (Leica).