Immunohistochemistry (IHC)

Tissue specimens were collected and processed as either formalin-fixed paraffin-embedded sections (FFPE) or cryosections. For FFPE sections, serial sections (4 µm) were cut and deparaffinized in 100% xylene, then rehydrated in a series of ethanol baths and washed with phosphate buffered saline (PBS). Slides were incubated in 3% H₂O₂ in methanol to quench endogenous peroxidases and blocked in TNB protein blocking solution (Thermo-Fisher Scientific, Waltham, MA). Primary antibody (podoplanin 1:25, Covance Laboratories, Dedham, MA) was incubated overnight at 4°C. The following day, sections were incubated in biotinylated secondary antibody (1:200, Vector Laboratories, Burlingame, CA) followed by alkaline phosphatase-conjugated avidin (Vectastain ABC-AP Universal Kit; Vector Laboratories, Burlingame, CA). Expression was visualized with the Vector Red chromogenic substrate kit (Vector Laboratories, Burlingame, CA) and counterstaining was performed using Gill no. 3 hematoxylin (Sigma-Aldrich, St. Louis, MO).

For frozen tissue, serial cryosections (8 – 10 µm) were cut and stored at −80°C prior to use. Slides were air-dried at room temperature (RT), fixed in 100% acetone, washed with PBS, incubated in 3% H₂O₂ in methanol, and blocked in TNB. Primary antibodies cluster of differentiation 31 (CD31 (1:200 Dako, Carpinteria, CA) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (1:200 ReliaTech GmbH, Wolfenbuttel, Germany) were added for two hours at RT. Sections were then incubated in biotinylated secondary antibody (1:200, Vector Laboratories, Burlingame, CA) followed by alkaline phosphatase-conjugated avidin. Vector Red chromogenic substrate kit was used for visualization followed by a counterstain with Gill no. 3 hematoxylin. All imaging was performed using an Axioskop 2 MOT Plus microscope (Carl Zeiss Inc., Thornwood, NY).