The use of immunohistochemical techniques has enhanced the diagnostic intensities in pathology. The sensitivity and specificity of any immunohistochemical protocol mainly depends on the ability to unmask antigen from formalin fixed tissue. Various combinations of antigen retrieval protocols have been used to unmask antigen efficiently but none has been established as a flawless one. These protocols either have one or the other lacunae, lacking repeatability under same conditions. In our study, we tried to develop a standardized immunohistochemical protocol for the identification of blood and lymphatic vessels in tissue sections of canine mammary tumour (CMT) using recently identified markers. The combined effects of antigen retrieval (AR) methods including pH shock by Heat-induced epitope retrieval (HIER) and Protease-induced epitope retrieval (PIER) were found highly effective in retrieval of tedious antigens of lymphangiogenesis and angiogenesis. The use of combined antigen retrieval technique for the unmasking of antigens from over fixed, old formalin fixed paraffin embedded (FFPE) canine mammary tumour sections resulted in efficient unmasking of epitopes when compared with the individual antigen retrieval methods.

Lymphangiogenesis and Lymphatic MarkersLymphangiogenesis is the formation of new lymphatics which facilitates cancer progression through a series of sequential processes that include dissemination and invasion into surrounding stromal tissues from a primary tumor, penetration into lymphatic walls and implantation in regional lymph nodes, extravasation,

Canine mammary tumour (CMT) is an excellent, naturally occurring animal model for human breast cancer. Most research, so far, has focused on angiogenesis as a mode of tumour invasion and metastasis, but the role of lymphangiogenesis in mammary tumours is scarcely documented. The present study was conducted on 45 cases of CMT and the extent of lymphangiogenesis, demonstrated as intratumoural vessel density (IVD) and peritumoural vessel density (PVD) was determined by immunohistochemical staining with lymphatic markers, viz. podoplanin, LYVE-1, PROX 1 and VEGFR-3. The highest vessel density in intratumoural area was recorded with VEGFR-3 (35.94 ± 3.45), followed by podoplanin (31.95 ± 2.77), LYVE-1 (11.11 ± 2.20) and PROX 1 (7.62 ± 1.11). In peritumoural areas, the vessel density was highest with podoplanin (11.48 ± 1.32), followed by VEGFR-3 (7.69 ± 0.51), LYVE-1 (5.19 ± 0.96) and PROX 1 (3.48 ± 0.48). The lymphatics in intratumoural areas were small, thin and collapsed, whereas the peritumoural lymphatics were large and conspicuous. Overall survival was less in patients with high lymphatic density in peritumoural areas for podoplanin, and with high vessel density in intratumoural areas for VEGFR-3. The survival was also low in cases with lymphatic tumour emboli. It was concluded that both intratumoural and peritumoural lymphangiogenesis seemed to play significant role in tumour invasion and spread rather than peritumoural lymphatics. Moreover, the podoplanin and PROX 1 were found to be more specific markers of lymphangiogenesis.