Topic Introduction

Fixation and Permeabilization of Cells and Tissues

Adapted from "Preparation of Cells and Tissues for Fluorescence Microscopy," Chapter 4, in Basic Methods in Microscopy (eds. Spector and Goldman). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

Fluorescence microscopy is used to visualize specific cellular components in as native a state and organization as possible. This article describes some of the main issues that must be considered when cells and tissues are fixed and permeabilized. To preserve cellular structure, the specimen is fixed chemically to retain the cells or tissue in a state as near to life as possible by rapidly terminating all enzymatic and other metabolic activities to minimize post-fixation changes. Sample fixation is one of the most crucial steps in assuring the accuracy of detection protocols and is therefore decisive in determining the subsequent success or failure of a given experiment. Underfixation of the sample leads to poor morphological preservation and/or loss of signal, whereas overfixation may lead to fixation artifacts, loss of signal, and/or increased nonspecific background signals ("noise"). An ideal fixative should preserve a given antigen in a fashion that reflects the in vivo situation with respect to its distribution (no diffusion or rearrangement). Ideally, cell morphology should be preserved, the antigen of interest should remain accessible to the probe, and the fixation should cause minimal denaturation of the antigen. However, several of these goals are mutually incompatible, and therefore, a compromise must be attained.