Capsure macro cap

Twenty core metastatic disease biopsies representing pre- and post-PIKTOR treatment specimens were embedded in Optimal Cutting Temperature (OCT) compound and sectioned (8 μm) onto plain glass microscope slides. Histomorphology was assessed with hematoxylin-eosin (H&E) staining following standard protocols [35 ].
Tumor and stromal cells were isolated as separate enriched cell populations using LCM (ArcturusXT or PixCell IIe instruments) in infrared capture mode as previously described [35 ]. Frozen tissue sections were fixed in 70% ethanol, stained with hematoxylin, dehydrated in graded ethanol and rinsed in xylene. The staining reagents contained protease inhibitors (CompleteMini tablets, Roche#11836153001). Microdissected cells captured on LCM caps (CapSure Macro cap, ThermoFisher #LCM0211) were lysed with protein extraction buffer (10% (v/v) Tris(2-carboxyethyl)phosphine (TCEP; Pierce #77720, Rockford, IL) in equal volumes of Tissue Protein Extraction Reagent (T-PER™, Pierce #78510) and Novex Tris-glycine 2X SDS buffer (Invitrogen #LC2676). Lysates were denatured at 95 °C for 5 min and stored at -80 °C prior to reverse phase protein array construction.

FFPE blocks were obtained from UCSD Health Anatomic Pathology after surgical biopsy, excision or mastectomy. The UCSD institutional review board approved the retrospective study and waived the requirement for consent. Consecutive sections of the blocks were used for Hematoxylin–Eosin staining (N = 1; 4 µM glass slide) then for Laser Capture Microdissection (LCM; N = 3; 7 µM glass slide coated with polyethylene naphthalate—ThermoFisher #LCM0522). The slides were stored at − 20 °C in an airtight container with desiccant until ready for dissection (1 day to 3 months). The LCM sections were thawed and stained with eosin, sections were kept in xylene and dissected within 2 h of staining. Laser Capture Microdissection was performed using the Arcturus Laser Capture Microdissection System. Matching regions from 3 adjacent sections were collected on one Capsure Macro Cap (Thermofisher), region size permitting. Post-dissection, all caps were covered and stored at − 20 °C.