Fixation methods paraformaldehyde

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

Today, for research with animal tissue and cell cultures, the standard has become fixation in paraformaldehyde, with animal tissue sectioned in a cryostat, and then incubation of sections and cultures with antibodies. This book focuses on introducing the methods of immunocytochemistry for biomedical scientists. These chapters may be read in order for a complete understanding of immunocytochemistry, or the chapters may be read individually for information about specific topics. The book is designed to help the novice perform experiments, solve problems, get results, and understand more advanced texts when more advice is needed. 

Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. I. Immunol. Methods. 1981 47 25-30. 

The IEM double-labeling method described was performed with cells fixed in 2% paraformaldehyde and 0 04% glutaraldehyde, and embedded in Lowicryl K4M, LR White, or LR Gold. Other fixation and embedding procedures should work equally well, provided that the specimen resists the chemicals used m the silver enhancement. 

The method of Pickel et al. (1976) is simple for large tissue fragments if the antigen withstands fixation. Organs or animals are perfused with a warm mixture of 1% GA and 1% paraformaldehyde in 100 mM sodium cacodylate buffer, pH 7.15. Microtome sections are pretreated with Triton X-100, normal serum and hypertonic buffer prior to immunostaining. 

Animal tissue can be fixed in situ by perfusion with 4% paraformaldehyde, followed by immersion fixation of the dissected tissue (1-4 h, depending on the tissue size and fixation obtained with perfusion). This method is strongly recommended if brain or spinal cord tissues are used, as these tissues do not fix well by immersion only, owing to the poor penetration of the fixative in the tissue matrix. 

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