Home › Cell Biology › Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue
Cell Biology JoVE (Open Access) Citable · DOI

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

DOI: 10.3791/60115-v
What you'll learn
  • ✓ Perform heat-induced epitope retrieval on paraffin-embedded tissue sections
  • ✓ Execute immunofluorescence staining protocol for NET detection
  • ✓ Identify and visualize neutrophil extracellular traps by microscopy
Protocol

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Difficulty
intermediate
Total time
~4–6 hours per tissue sample (including antigen retrieval and staining incubations)
Model organism
Mouse; Human tissue
Biosafety
BSL-1

Steps

1
Rehydrate and retrieve epitopes in tissue

Rehydrate paraffin-embedded tissue sections and perform heat-induced epitope retrieval to expose NET antigen binding sites. This step ensures optimal immunofluorescence labeling of NET components.

▶ 00:52
2
Stain tissue with immunofluorescence markers

Apply primary and secondary antibodies to label NET components (e.g., neutrophil elastase, DNA), then mount slides for visualization. Incubation times and antibody concentrations should be optimized per protocol.

▶ 03:57
3
Analyze NET structures by fluorescence microscopy

Acquire and examine immunofluorescence images to identify characteristic NET morphology and co-localization of neutrophil and extracellular markers in tissue samples.

▶ 06:16
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