Pitfalls and Caveats in Histochemically Demonstrating Apoptosis

Abstract

For investigating the biological significance of apoptosis, the exact and sensitive histochemical identification of apoptosis and apoptotic cells is essential. However, we need to recognize both the pitfalls and caveats in performing histochemical staining and in interpreting the findings obtained. DNA fragmentation-based approaches, such as TdTmediated dUTP-biotin nick end-labeling (TUNEL), in situ nick translation (ISNT) and immunostaining for single-stranded DNA, represent DNA alterations in the apoptotic cell, but they are technically unstable and occasionally give false positive and false negative findings. In contrast, immunostaining for intracellular proteins cleaved and activated by caspases, including cleaved caspase 3, cleaved poly(ADP-ribose) polymerase, cleaved cytokeratin 18 and cleaved actin (fractin), is technically reproducible, but the intracellular accumulation of the activated proteins is not necessarily synchronized. The present review focuses on the pretreatments for enhancing the sensitivity of these techniques, as well as their limitations and comparisons in histochemically demonstrating apoptosis and apoptotic cells.

Enzyme-labeled Antigen Method

“…Appropriate concentrations of the probe antigen solutions were also examined: 1-100 mg/ml for HRP, 10-100 mg/ml for biotinylated OA, and 100-500 mg/ml for biotinylated KLH. Heat-assisted retrieval through pressure pan cooking in 0.01 M citrate buffer, pH 6.0 or pH 7.0, and in 1 mM EDTA solution, pH 8.0, was also tried (Tsutsumi and Kamoshida 2003). The other procedures were fundamentally the same as in the case of frozen sections.…”

Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method

“…For retrieving the antigen-binding activity, the sections were pretreated with high concentration (20 μg/ml) of proteinase K at room temperature for 15 min. Heat-assisted retrieval by pressure pan cooking in 10 mmol/l citrate buffer, pH 6.0 or pH 7.0, and in 1 mmol/l EDTA solution, pH 8.0, was also tried (Tsutsumi and Kamoshida, 2003). The crude biotinylated protein solutions were incubated overnight at room temperature.…”

Association of Protein Phosphatase 1 Delta with Nucleolin in Osteoblastic Cells and Cleavage of Nucleolin in Apoptosis-induced Osteoblastic Cells

“…It was reported that morphological changes in apoptotic cells are associated with the alterations of various nuclear, cytoplasmic, and cell membrane proteins [4,19,29,47,64,67]. To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in AgNOR proteins and nucleolin during apoptosis in cultured cells [31,42].…”