Detection of apoptotic cells using immunohistochemistry

Area of Science:

• Oncology
• Cell Biology
• Immunohistochemistry

Background:

• Immunohistochemistry is a standard method for detecting apoptotic cells in situ.
• Apoptosis, or programmed cell death, is crucial in regulating cell populations and is often dysregulated in diseases like cancer.
• B-cell lymphoma is a type of cancer originating from B-cells, a type of white blood cell.

Purpose of the Study:

• To describe a detailed protocol for identifying apoptotic B-cell lymphoma cells in mouse models.
• To outline the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay combined with immunohistochemistry for apoptosis detection.
• To highlight the importance of confirmatory assays to ensure accurate results.

Main Methods:

• B-cell lymphoma cells were xenografted into recipient mice.
• Tumor-bearing mice were treated with vorinostat, a histone deacetylase inhibitor known to induce apoptosis.
• Tumor samples underwent fixation, sectioning, and DNA end-labeling using the TUNEL assay.
• Immunohistochemistry was employed to detect TUNEL-labeled DNA within B-cell lymphoma cells.

Main Results:

• The protocol successfully identified B-cell lymphoma cells undergoing apoptosis.
• Fragmented DNA, a hallmark of apoptosis, was detected in tumor cells post-treatment.
• The TUNEL assay combined with immunohistochemistry allowed for the visualization of apoptotic cells in situ.

Conclusions:

• The described method provides a reliable approach for detecting apoptosis in B-cell lymphoma xenografts.
• Confirmation of apoptosis using additional markers, such as active caspase-3 immunohistochemistry, is crucial to mitigate potential false-positive results from the TUNEL assay.
• This protocol can be valuable for researchers studying apoptosis in B-cell lymphomas and evaluating the efficacy of apoptosis-inducing agents.