Immunofluorescent staining tissue sections

MICROWAVE HEAT-ASSISTED IMMUNOFLUORESCENCE STAINING OF TISSUE SECTIONS... 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

Figure 2 Identification of adult cardiomyocytes with mitotic features. Immunofluorescence staining of transverse tissue sections from normal adult rat heart ventricles for pbosphorylatcd histone 3 (bright green), striated muscle myosin (red), and countcrstained for DNA (Hoechsst 33342, blue). Presence of bright green nuclei, surrounded by red cytoplasmic staining, identifies cardiomyocytes with mitotic features (thick arrows), while lack of red staining identifies non-myocytes (thin arrows).

Immunofluorescence staining permits detection of protein antigens in situ, in order to investigate the subcellular localization or cellular distribution within a tissue. The cells or tissue sections are fixed and incubated with the specific primary monoclonal antibody. The antigen-primaiy monoclonal antibody complex is bound by a second antibody conjugated to a fluorescent dye, such as rhodamine-p-isothiocyanate or fluorescein isothiocyanate, for detection by fluorescence microscopy. Immunofluorescence staining is described in more detail in Chapter 16. 

In this method, samples of freshly excised tissues were fixed by boiling them in a 10% neutral formaldehyde solution for 5 min. The fixed tissues were then embedded in paraffin and cut into 3 pm thick sections. The sections on glass slides were deparaffinized and dehydrated by passage through xylene and a graded alcohol series. Then they were incubated with a primary antibody against poly(ADP-ribose). The antibody used, IgG monoclonal antibody lOH, recognized the linear structure of poly(ADP-ribose) described previously (3). Bound antibody was stained with die avidin biotin peroxidase complex or by an indirect immunofluorescence technique. 

---