Immunohistochemical Evaluation of SRSF1 in MPM

Formalin-fixed and paraffin-embedded specimens were cut to 4–5 μm, mounted on sialinate-coated slides (Dako, Glostrup, Denmark), stored at room temperature and stained with hematoxylin and eosin. Pathological diagnosis of MPM was rendered according to WHO criteria. In addition, for each case, immunohistochemical investigation was carried out using antibodies anti-SRSF1 (sc-33652; working dilution 1:50; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-CD31 (JC70A; working dilution 1:40; DAKO, Glostrup, Denmark).
The detection of brown chromogen within tumor nuclei was considered as positive SRSF1 immunostaining; unaffected gallbladder tissue was adopted as a positive control (Figure 1), while negative control slides were obtained by incubating them with phosphate-buffered saline (PBS) instead of the primary antibody. A semi-quantitative analysis of the cases stained with SRSF1 was performed, as previously described [25 (link),26 (link),27 (link)]: briefly, the immunoreactivity score (IRS) was obtained by multiplying the intensity of staining (IS) and the percentage of positive cells (extent score; ES): if the IRS was ≤6, the SRSF1 expression was considered to be “low” (L-IRS), while an IRS > 6 was considered to be “high” expression (H-IRS).