Immunostaining of cell preparations: a comparative evaluation of common fixatives and protocols

Area of Science:

• Cytopathology
• Immunohistochemistry
• Biomedical research

Background:

• Immunostaining in cytology faces challenges with inconsistent results, high background staining, and antigen-specific fixative requirements.
• A universal fixation method is needed for reliable cytologic diagnosis across diverse tissue antigens.

Purpose of the Study:

• To identify a universal fixative and a simple protocol for preserving immunoreactivity of various tissue antigens in cytologic preparations.
• To overcome limitations of existing immunostaining fixation methods.

Main Methods:

• Evaluated 23 fixation protocols using various agents including acetone, ethanol, methanol, glutaraldehyde, and formal saline.
• Optimized fixation involved overnight 0.1% formal saline at 27°C followed by 10 min in 100% ethanol.
• Assessed immunoreactivity, background staining, morphology, and antigen preservation.

Main Results:

• Fixation in 0.1% formal saline followed by ethanol yielded optimal and consistent immunoreactivity for a wide range of antigens.
• This protocol eliminated the need for endogenous peroxidase blocking.
• Air-dried smears (≥14 hr) did not require adhesive slides and maintained immunoreactivity for up to 7 days at room temperature or 5 weeks at -70°C.

Conclusions:

• A universal fixation protocol using 0.1% formal saline and ethanol is effective for immunostaining cytologic preparations.
• This method enhances immunoreactivity, reduces background staining, and offers flexibility in sample storage.
• The protocol simplifies cytologic diagnosis by providing consistent results across multiple antigens.