Protocol

Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Indirect Immunofluorescence

This protocol was adapted from "Flow Cytometry," Chapter 16, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.

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INTRODUCTION

This protocol provides a method for analysis of cell surface antigens using indirect immunofluorescence, i.e., when both a primary and secondary antibody are being used to produce fluorescence. After staining, the cells can be analyzed immediately by flow cytometry or fixed and stored for later analysis.

MATERIALS

Reagents

Antibodies, primary and secondary

BSA or heat-inactivated serum

EDTA

EGTA

Paraformaldehyde in PBS, 2% (Optional, see Step 16)

PBS, prepared without calcium and magnesium

Sodium azide (Optional, see Step 2)

METHOD

Proper controls are essential in flow analysis. The first control performed should be autofluorescence; for this control, the sample is carried through the protocol . . . [Full Text of this Article]