Harris haematoxylin

Boyden chambers (8μm pores) were placed into 24-well plates containing normal growth media and 10ng/ml EGF, as a chemo attractant. MCF7-LTED and MCF7-TAMR cells were pre-treated with 100nM dasatinib for 1-hour and then seeded at a density of 5x10⁴ in the upper chamber in serum free media for 48-hours. Non-migrating cells were scraped from the top surface of the membrane while migrated cells were stained with Harris’ Haematoxylin (VWR). The membranes were excised and mounted on a microscope slide and the number of migrating cells counted.

Rehydrated sections were treated with 1% H₂O₂ to block endogenous peroxidase activity, followed by citrate treatment as described above. Sections were washed with the addition of 0.1% Tween to the PBS solution, were blocked with 10% normal horse serum and incubated overnight with anti‐APP (Invitrogen 13–0200, 1:100) at 4°C. Sections were then incubated with biotinylated horse antimouse (1:200) for 45 min. The antigen was visualized as above. Following this, sections were then counterstained in Harris' haematoxylin (VWR, Dublin, Ireland) and dehydrated before coverslipping with DPX.
Immunohistochemically labelled sections were captured at 40×. Four sections of PrTN from 3–4 animals from each group were analysed as follows: pictures were processed to remove the Harris' haematoxylin counterstaining using the separate blue on image tool of Cell A software and saved as a new picture; the resulting pictures were opened in ImageJ and thresholded such that only APP‐positive labelled spheroids had sufficient transmittance to be retained. Spheroids were then assessed using the ‘analyse particles’ function using predefined parameters (size: 5–500 and circularity: 0.7–1). The mask tool allowed depiction of the selected particles for analysis. All samples were photographed on the same light and colour settings to minimize any influences of microscopy and camera settings.

Tumour cells were isolated using LMD (Leica LMD6000, Leica Microsystem). Briefly, frozen tumour tissue was embedded in optimal cutting temperature (OCT) compound (Leica Microsystems, Wetzlar, Germany), cut into 20 µm thick sections using Leica CM1850 UV Cryostat (Leica Microsystems) at − 20 °C and mounted onto PET-membrane steel frame slides (Leica Microsystems). The sectioned tissues were fixed with 70% ethanol for 30 s, and stained in Harris’ haematoxylin (VWR, England) for 30 s then rinsed in water and air dried. All staining procedures were carried out on ice with all solutions supplemented with fresh 1 mM phenylmethanesulfonyl fluoride (Sigma, St Louis, MO, USA). The estimated total protein yield from one thousand cells was approximately one µg protein.