Ab87322

A total of 37 formalin-fixed, paraffin-embedded tissues were obtained from patients diagnosed with SCLC by bronchofiberscopy or biopsy between January 2012 and March 2015. All patients received care and follow-up in our hospital. Informed consent was obtained under the protocol approved by our hospital's ethics committee. The clinical characteristics are summarized in Table 1.
The endogenous peroxidase activity was blocked by soaking the deparaffinized specimens in 3.3% H₂O₂. The specimens were then incubated with primary antibodies (MST2, 1 : 200, ab87322, abcam; pMST2 (phosphor Thr180), 1 : 200, PA5-104616, Invitrogen; YAP1, 1 : 100, ab205270, abcam; pYAP1 (phosphor S127), 1 : 100,ab76252, abcam; CD133, 1 : 100, ab216323, Abcam) and the corresponding secondary antibodies [20 (link)]. Semiquantitative results were obtained by using the German semiquantitative scoring method, as previously described [21 (link)].

We selected typical sections of haematoxylin and eosin-stained slides that represent prominent intratumoral regions among the collected samples. From the invasive tumour front of each representative paraffin block, single core was obtained and transferred to a recipient TMA block.
Six types of immunostaining were performed on TMA using recommended doses of six different reagents according to the corresponding manufacturer’s instructions. Phospho-YAP (Ser127) (1:1000, monoclonal, D9W2I, #13008; Cell Signaling, Danvers, MA, USA), YAP (1:200, monoclonal, D8H1X, #14074; Cell Signaling, Danvers, MA, USA), KIBRA (1:200, polyclonal, ab216508; Abcam, Cambridge, MA, USA), LATS1/2 (1:200, polyclonal, #PA5-115498; Invitrogen, Carlsbad, CA, USA), Merlin (1:250, polyclonal, ab217016; Abcam, Cambridge, MA, USA), and MST1/2 (1:250, polyclonal, ab87322; Abcam, Cambridge, MA, USA) were used as primary antibodies. p-YAP, YAP, KIBRA, Merlin, and MST1/2 showed a diffuse staining pattern in the cytoplasm of cancer cells, whereas LATS1/2 showed a focal staining pattern in the cell nucleus. The staining level was read as 2-tiered. The stained tumour cells were graded as either positive or negative based on a higher intensity than that found in stromal cells and lymphocytes and an intensity equal to or lower than that of non-tumour cells, respectively.