Formalin fixation and paraffin embedding

Morgan JM. A protocol for preparing cell suspensions with formalin fixation and paraffin embedding which minimises the formation of cell aggregates. J Cell. Pathol. 2001 5 171-180. 

Antigenic determinants masked by formalin-fixation and paraffin-embedding may also be exposed by enzymatic digestion. This can, however, not be used with frozen sections or cells which are not paraffin-embedded. The beneficial effects of protease treatment are presumably related to cleavage of the molecular cross-links by the... 

The usual method of sample preparation for tissue remains as formalin fixation and paraffin embedment (FFPE). This venerable approach may be satisfactory for the preservation of morphologic detail, but does adversely affect the antigenicity of many target molecules in the tissue, to degrees that are unknown. The enormous variation in protocols (including fixation times) employed for FFPE among different laboratories, or within the same laboratory from specimen to specimen, compounds the problem, and contributes to the current poor reproducibility. 

In Table 5.1 a summary of the main findings from section 2 is presented. The main FTIR and Raman bands affected by tissue drying, cryopreservation, formalin fixation and paraffin embedding are listed as these are the standard tissue processing methods used for histopathology. 

Castiglione F, Degl Innocenti DR, Taddei A, et al. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues effects of the fixation on outcome reliability. Appl. Immunohistochem. Mol. Morphol. 2007 15 338-342. 

The protocols described below illustrate (1) frozen section sample preparation, (2) hematoxylin and eosin (H E) tissue staining, and (3) automated LCM. Alternative tissue preparation methods, such as ethanol or formalin fixation with paraffin embedding, are acceptable for RNA and DNA analysis... 

Those interested in using immunohistochemistry to study apoptosis need to consider the method of tissue preparation and fixation. Since many antibody epitopes do not survive formalin/glutaraldehyde fixation or paraffin embedding, investigators should determine under what conditions the antibody of interest will work prior to sample collection. There are antibodies that will successfully bind to formalin-fixed, paraffin-embedded material, but if the investigator is unsure, fresh snap-frozen samples can be used to optimize conditions for success since freezing generally will not alter epitopes. 

Vollmer E, Muller AM, Muller-Navia J (2001) HOPE fixation a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 197 823-826 7. Werner M, Chott A, Fabiano A, Battifora H (2000) Effect of formalin tissue fixation and... 

A simple and effective AR technique of boiling archival paraffin-embedded tissue sections in water to enhance the signal of IHC was developed to circumvent the deleterious effects of formalin fixation, which had previously... 

Figure 5.1 Diagrammatic explanation of standardization of IHC via AR and test battery to achieve a maximal retrieval level by an optimal protocol of AR. The intensity of IHC (axis y) is inversely correlated with the time of formalin fixation (axis x) as indicated by a reduced slope. Three arrows indicate a potential maximal retrieval level that may equalize the intensity of IHC to a comparable result for routinely processed, paraffin-embedded tissues with various time of fixation. Reproduced with permission from Shi et alHistotechnol. 1999 22 177-192.

In 1991 Shi et al. published their seminal observation that high-temperature incubation of formalin-fixed, paraffin-embedded (FFPE) tissue sections in buffers for short periods led to improved immunohistochemical staining.1 However, more than 15 years later, heat-induced antigen retrieval (AR) remains largely an empirical procedure, requiring the optimization of several critical parameters by trial and error.2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method. 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

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