In totum immunostaining: a histological analysis tool for small dimensions biological samples

Abstract

Immunostaining through immunohistochemical or immunofluorescence techniques has been widely used in recent years as a result of the increasing availability of antibodies. The identification of specific epitopes in biological tissue by antigen-antibody reactions, detected by a visual marker, that allows their identification under microscopy, provides more accurate diagnoses. Today, the most commonly used methods in immunohistochemistry refer to histological sections obtained from paraffin sections, cryostat or cell suspensions. However, these methodologies are not always suitable, especially for small samples. Thus, in order to perform immunostaining in difficult to manipulate biological tissues, in totum immunohistochemical processing was performed in embryos, larvae and total biological tissues of fishes. The development of this technique was adapted from proposed protocols for free-floating immunohistochemistry. The in totum biological samples immersed in different types of fixing solutions and washed in Sorensen Phosphate Buffer. Next, endogenous peroxidase was blocked. The antigen recovery was performed, followed by the nonspecific blockade and incubation with different primary antibodies. Subsequently, the in totum samples were incubated with different biotinylated secondary antibodies and stained with DAB-H2O2. The material was washed in distilled water and fixed again. Fluorescent secondary antibodies were also used in other similar samples, following the same initial protocol. The samples were submitted to historesin embeding and the histological sections (5μm thick) were obtained in microtome. DAB stained immunohistochemistry slides were counterstained with Hematoxylin. Samples fluorophore labeled were imunolabeling with DAPI. The immunostaining in both immunohistochemistry and immunofluorescence was maintained throughout historesin processing. The historesin embedding allows a better preservation of biological tissues and obtaining thinner histological sections that paraffin or frozen material. Consequently, the image resolution is potentially improved. In this regard, this work becomes an important toll to be used in immunohistochemical techniques, giving better results in tissues that are difficult to process in paraffin or freezing