Superdab

Samples were fixed in 4% paraformaldehyde (PFA) overnight prior to standard processing for paraffin embedding. Serial paraffin sections were cut at 5 μm thickness. Haematoxylin and eosin, Periodic acid-Schiff, picrosirius red and Masson’s trichrome staining were performed on sections using an automated staining system (Tissue-Tek). For immunofluorescence staining, slides were dewaxed using an automated protocol and antigen retrieval was performed using citrate buffer (pH 6.0). Primary antibodies against cytokeratin 5 (CK5; Abcam; ab17130), collagen IV (COL-94; Abcam; ab6311) and laminin (Sigma; L9393) were used. Species-appropriate secondary antibodies conjugated to AlexaFluor dyes (Molecular Probes) were then used. Images were acquired using a Zeiss LSM700 confocal microscope. For immunohistochemistry, slides were dewaxed and antigen retrieved as for immunofluorescence. Primary antibodies against CD45 (Abcam; ab10558), endomucin (V.7C7.1; Abcam; ab106100) and F4/80 (Serotec; MCA497GA) were used. Sections were then incubated with species-appropriate biotinylated F(ab’)2 for 30 min, developed using ABC reagent and superDAB (Dako) and finally counterstained with haematoxylin.

Breast tissue samples were obtained from surgical specimens from patients undergoing breast surgery at Barts Health NHS Trust London. The study was performed following patient consent and approval from the local research ethics committee (reference: 05/Q0403/199 and 09/H075/39). Seven were normal breast cases from reduction mammoplasty, nine were cases of DCIS (high, intermediate and low grade) and nine were cases of DCIS with concomitant invasion.
Sections were dewaxed in xylene and antigen retrieved in citrate buffer pH6; followed by incubation with MMP-8 rabbit polyclonal antibody (Atlas, Cambridge, MA, USA, HPA02122, 1:750). Sections subsequently were incubated with goat anti-rabbit biotinylated F(ab’)2 for 30 min, developed using ABC reagent and superDAB (Dako, Glostrup, Denmark) then counterstained with hematoxylin [7 (link)].