S3003

Scaffolds with fragments of surrounding tissues were fixed for 24 h in Bouin’s acidic solution (1.3% trinitrophenol (Sigma-Aldrich, St. Louis, MO, USA), 40% formalin (hereinafter BioVitrum, unless otherwise indicated)). After washing in 70% ethanol, standard histological preparation of samples was performed, after which they were placed in paraffin medium (Histomix, Little Rock, AR, USA). Paraffin sections 5 µm thick were obtained using a microtome (Leica RM2235, Miami, FL, USA) and placed on silanized slides (S3003, Dako). Deparaffinized sections were stained with hematoxylin and eosin (8GX, Sigma-Aldrich, St. Louis, MO, USA). Sections were dehydrated in alcohol, cleaned with (ortho-)xylene, and embedded with Canadian balsam (Merck, Boston, MA, USA). Histological sections were examined under an AXIO Imager A1 microscope (Carl Zeiss, Jena, Germany) with a Canon Power Shot A640 (Canon, Ohta-ku, Tokyo, Japan) camera.

CLs were fixed with 4% (v/v) paraformaldehyde (PFA) in PBS. 4 µm sections were mounted on glass slides pre-coated with silane (S3003; Dako, Glostrup, Denmark), deparaffined and rehydrated. Antigen retrieval was achieved by Tris-EDTA buffer pH 9.0 using microwave for 15 min at 600 W. Sections were immersed in methanol with 3% (v/v) H2O2 for 30 min and incubated with 10% (v/v) normal horse serum (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for blocking. Then, sections were incubated with LYVE-1 antibody (ab33682; abcam, Cambridge, UK) as a specific marker of lymphatic endothelial cells [10] (link), [15] (link), [16] (link) diluted at 1∶4000 with PBS for 1 h at room temperature (RT). Negative control sections were incubated with normal rabbit serum diluted by PBS. Subsequently, sections were incubated with ImmPRESS UNIVERSAL reagent, anti-mouse/rabbit Ig (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 30 min according to the manufacturer’s instructions. The staining was visualized with 0.05% (w/v) 3,3′-diaminobenzidine (343-00901; Dojindo, Kumamoto, Japan) containing 0.01% (v/v) H2O2 and then counter-stained with hematoxylin. Bright field images were captured using FSX100 (Olympus, Tokyo, Japan) and merged using cellSens (Olympus, Tokyo, Japan).