Abstract
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.
Highlights
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
The use of single retrieval buffer, most commonly recommended for carrying out immunohistochemistry i.e. citrate buffer did not yielded any significant results in antigen unmasking and most of the sections as stained with the antibodies directed against podoplanin, LYVE-1, PROX 1, VEGFR-3, VEGFR-2, laminin, VEGF-D and VEGF-C were either negative or mild in intensity
The biggest challenge of IHC is to develop standardized methods that perfectly reverse the conformational changes produced during aldehyde fixation and approximately 85% of Antigens fixed in formalin require some type of antigen retrieval (AR) to optimize the immunoreactions [26] [27]
Summary
Growing interest in immunohistochemical procedure has led to the develop-. K. Sood ment of a wide range of highly specific immuno-stains, offering a greater role in pathological investigative studies [1]. The biggest deficiency in immunohistochemistry has been how to maintain both good morphology and the immuno-reactivity of antigens in tissue sections. Various techniques of unmasking and retrieving the antigens from tissue section have revolutionized the patho-clinical diagnosis and have played a pivotal role in enabling immunohistochemistry as a major diagnostic tools in case of formalin-fixed, paraffin-embedded tissues [2]. Antigen Retrieval (AR) techniques are aimed at significantly increasing the sensitivity of the immunohistochemical detection of epitopes in addition to better morphologic details and more precise antigen localization [3] [4]
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