Anti srsf1 sc 33652

Tumors were fixed in 4% paraformaldehyde immediately after removal from nude mice. After tissue embedding, slicing, dewaxing, and incubating with antibodies, images were taken at the proper magnification using a microscope (Leica Microsystems, Mannheim, Germany). Antibodies used in this experiment were, anti-PCNA (10205-2-AP, Proteintech, USA), anti-SRSF1(sc-33652, Santa Cruz Biotechnology, China) and anti-β-catenin (51067-2-AP, Proteintech, USA).

Protein samples were mixed 1:1 with 2× Laemmli buffer (Sigma, UK) and incubated at 100 °C for 5 min before being chilled on ice. Protein samples of equal amounts were subjected to SDS-PAGE and transferred to PDVF membranes. Membranes were incubated at 4 °C overnight with one of the following antibodies; anti-CA IX (M75 monoclonal antibody, Novus Biologicals); anti-SRSF1 (sc-33652, SantaCruz Biotechnology, UK); anti-CLK1 (R1471–1 s, Abiocode, USA); anti-phospho SR(1H4) (sc-13509, SantaCruz Biotechnology, UK); anti-β-actin (ab8226, Abcam, UK). Horse anti-mouse IgG HRP-linked antibody (7076S, Cell Signalling, UK) was used to detect all antibodies apart from anti-CLK1, which was detected by goat anti-rabbit IgG HRP-linked antibody (7074, Cell Signalling, UK). Laminata Forta Western HRP substrate (Millipore, UK) was used to image protein bands.

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).