Superfrost plus

For immunofluorescence (IF) analyses on bioptic samples, serial cryostat sections (7 μm) were collected on positively charged slides (BDH Superfrost Plus), air dried at room temperature (RT) and fixed with PFA for 15 min. After washing with phosphate-buffered saline (PBS), the slices were incubated with 0.5% Triton X-100 in PBS for 5 minutes at RT, saturated with 1% BSA−10% normal goat serum (DAKO) in PBS for 1 h at RT, and stained ON at 4 °C with the appropriate antibodies. Next, the samples were washed with PBS and incubated with the appropriate secondary antibodies and TO-PRO3 to stain the nuclei for 1 hour at RT. After washing with PBS, the slides were mounted in Mowiol containing 2.5% (w/v) of 1,4-diazabicyclo-(2,2,2)-octane (DABCO). Images were acquired with a Leica TCS SP8 Confocal system (Leica Microsystems Heidelberg, Mannheim, Germany), using the Leica Confocal Software (LCS). The monoclonal anti-human CD31, CD34, and CD105 antibodies were from Abcam (Cambridge, UK). The polyclonal anti-human Multimerin-2 (MMRN2) antibody was produced in our laboratories as previously described (26 (link)); the polyclonal anti-human GLUT 1 antibody was from Millipore (Temecula, CA, USA). The secondary antibodies conjugated with Alexa Fluor 488, 546 and TO-PRO-3 were from Invitrogen (Milan, Italy).

Fourteen tissue microarrays (TMAs) with primary breast cancer were prepared as described before [43 (link), 44 (link)]. Briefly, each TMA comprised of 0.6- (Hamburg Cohort, TMA n = 3) or 1.5-mm (Polish Cohort, TMA n = 11) diameter tissue cores obtained from formalin-fixed paraffin embedded breast cancer specimens. Cores of normal breast, colon, pancreas or tonsil tissues were introduced to TMAs as internal controls. Patients were represented by one (n = 355) or two (n = 243) fragment(s) of tumor (TMA tissue cores). One-hundred-ninety-nine fragments of breast cancer lymph node metastases (LNM) from 164 selected patients (1 to 4 samples per patient) were embedded into primary tumor TMAs (Hamburg Cohort) or constructed as 3 additional TMAs (Polish Cohort). TMA sections 4–6 μm thick were placed on charged polylysine-coated slides (Superfrost Plus, BDH, Germany) for further examination.

For immunofluorescence analyses on tumor samples, serial cryostatic sections (7 μm) were collected on positively charged slides (BDH Superfrost Plus), air dried at room temperature (RT) and fixed with PFA for 15 min. After washing with phosphate-buffered saline (PBS), the slices were incubated with 0.5% Triton X-100 in PBS for 5 min at RT, saturated with 1% BSA, 10% normal goat serum (DAKO) in PBS for 1 h at RT, and stained ON at 4 °C with the appropriate antibodies. Next, the samples were washed with PBS and incubated with the appropriate secondary antibodies and TO-PRO3 to stain the nuclei for 1 h at RT. After washing with PBS, the slides were mounted in Mowiol containing 2.5% (w/v) of 1,4-diazabicyclo-(2,2,2)-octane (DABCO). Images were acquired with a Leica TCS SP8 Confocal system (Leica Microsystems Heidelberg, Mannheim, Germany), using the Leica Confocal Software (LCS). Fluorescence intensity and quantification was evaluated by means of the Volocity software (PerkinElmer Inc., Waltham, MA, USA).