Cellular immunofluorescence: quantification of low abundance proteins

Area of Science:

• Cell biology
• Immunofluorescence techniques
• Biochemistry

Background:

• Indirect immunofluorescence is a common technique for visualizing cellular antigens.
• Quantifying antigen levels in immunofluorescence microscopy has been challenging.
• Fluorescein is a widely used fluorochrome in biological imaging.

Purpose of the Study:

• To develop a simple and accurate method for quantifying cellular antigens stained with fluorescein-conjugated antibodies.
• To enable the measurement of antigen levels in fixed cells used for immunofluorescence microscopy.
• To allow for normalization of antigen quantity to DNA content or cell number.

Main Methods:

• Extraction of aminofluorescein from fixed cells using an alkaline carbonate buffer.
• Quantification of extracted aminofluorescein via spectrofluorometry.
• Creation of a standard curve using known concentrations of fluorochrome for accurate analysis.

Main Results:

• A reliable method for extracting and quantifying aminofluorescein from stained cells was established.
• Spectrofluorometry allowed for precise measurement of fluorescence intensity.
• The method demonstrated the ability to quantify antigen levels in immunofluorescence samples.

Conclusions:

• The described method provides a straightforward approach to quantify cellular antigens in immunofluorescence microscopy.
• This technique can be adapted to determine DNA content, enabling antigen normalization.
• The quantification of antigens relative to DNA or cell number enhances the reliability of immunofluorescence data.