Tissue preparation for immunocytochemistry.

Abstract

Aims-To investigate the effect of tissue preparation on immunostaining and to establish whether there is a standard tissue preparation schedule that allows optimal demonstration of all antigens. Methods-Blocks of tonsil were subjected to variations to a standard fixation, processing, and section preparation schedule. The sections were stained with five antibodies-L26 (CD20), UCHL1 (CD45RO), CD3, vimentin, and antikappa light chain-using the streptavidinbiotin immunostaining technique. When further investigation was necessary, other tissues and antibodies were used and where weak immunostaining was obtained the use of microwave pretreatment to improve staining was tested. Results-Several factors involved in fixation were found to affect immunoreactivity. These included the duration, pH, and type of fixative used. In tissue processing only temperature and the duration of the dehydration and wax infiltration steps affected immunoreactivity. Of all the factors investigated, the temperature and duration of the section drying had the greatest effect. In contrast, long term storage of cut sections before immunostaining had no effect on the reactivity of the antibodies tested. Antibodies were found to be affected by alterations to tissue preparation by varying degrees, UCHL1 and vimentin being the most susceptible to changes in fixation and L26 to changes in processing. Where weak staining occurred, microwave pretreatment was generally found to eliminate-the problem. Conclusions-There is no standard tissue preparation schedule for the optimal demonstration of all antigens. Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed". ( Clin Pathol 1997;50:422-428)

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation

“…Loss of immunoreactivity in underfixed tissue (Figure 7a and Table 3) is probably caused by alcohol precipitating of unfixed tissue proteins, resulting in excessive sensitivity to proteolytic enzyme or microwave antigen retrieval pretreatment. 3,5,38 Tissues with 6-h conventional formalin fixation showed a stronger immunohistochemistry signal after antigen retrieval treatment than tissues with 22-h fixation, suggesting that the latter produces overfixation that is difficult to reverse fully by antigen retrieval.…”

Immunohistochemical Detection of Nestin in Pediatric Brain Tumors

“…Improved detection of several intermediate filament proteins in formalin-fixed brain tissue has been reported, including phosphorylated and non-phosphorylated NF (Evers andUylings 1996), vimentin (Ashraf Imam et al 1995;Shi et al 1991;Williams et al 1997) and GFAP (McQuaid et al 1995) and, when compared to protease digestion, MW pretreatment was clearly superior with antigens such as vimentin (Shi et al 1991). Antigen retrieval IHC was used to study nestin expression in developing and adult rodent tissue by use of the mouse MAb Rat-401 (Mokry and Nemecek 1998).…”

Decreased expression of BATF2 is associated with a poor prognosis in hepatocellular carcinoma

“…Finally, the visualization signal was developed with diaminobenzidine, and all the slides were counterstained with hematoxylin. As a positive control, Vimentin (mouse anti-Vimentin, sc-32322, Santa Cruz Biotechnology, Santa Cruz, CA, at a 1:200 dilution), a normally expressed protein that is reported to be the most susceptible to the changes of fixation, 5,6 was chosen to exclude the influence of tissue processing. As a negative control, tissue sections were immunoreacted with PBS under the same experimental conditions.…”