Histostain plus broad spectrum kit

For histology, tissues were fixed in the 10% formalin, blocked in paraffin, sectioned as 5 µm thick, stained with hematoxylin and eosin, and examined by light microscopy. Antibodies against ERalpha (sc-542; Santa Cruz), PR (sc-166169; Santa Cruz), ERBB2 (sc-33684; Santa Cruz), and MRC2 (sc-271148; Santa Cruz) were used for immunohistochemical staining, by using a Histostain® Plus Broad Spectrum kit (859043; Life Technologies) as per the manufacturer's instruction. Antibodies against Keratin14 (PRB-155P; Covance), Keratin18 (sc-53256; Santa Cruz), E-Cadherin (3195; Cell Signaling Technology) and Vimentin (5741; Cell Signaling Technology) were used for immunoflouresencent staining, and nuclei were stained with DAPI (62248; ThermoFisher Scientific).

The harvested liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 3-μm sections. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemical (IHC) staining, after deparaffinization, rehydration, and antigen retrieval, 3% H₂O₂ was used to inactivate endogenous peroxidases, and goat serum was used to block nonspecific binding sites. Then, the sections were incubated with appropriate primary antibodies overnight at 4 °C. Subsequently, the Histostain-Plus BroadSpectrum Kit (Life Technologies, USA, 859043) was used for staining. Then, the sections were counterstained with hematoxylin. The number of positive cells was determined in high-power fields (200 ×) of each liver tissue section. The utilized primary antibodies are listed in Additional file 1: Table S3.

The MPM tissue slides from the patient with multiple primary cancers were used for IHC staining with anti-calretinin antibody (# 180211, ThermoFisher) as previously described [34 ]. Briefly, 5μm-thick slide sections were deparaffinized with xylene and steamed in citrate for 20 minutes. They were then treated with blocking solution, washed, and incubated overnight. The slides were washed again and incubated with solutions from the Invitrogen Histostain Plus Broad Spectrum Kit (85-9643), washed again, stained with hematoxylin for 1 minute, mounted, and analyzed.