Bouins fixative

A marked degree of variability in the immunohistochemical results has been demonstrated in different breast cancer studies (56-58). The degree of positivity varied from 15.5-54% in 14 breast cancer studies (57). The number of immunohistochemically positive cells varied from 29-54% with four different p53 antibodies (59). Dissimilar fixation procedures and different paraffin temperature may provide explanation for the partly different immunohistochemical results (60). The importance of these issues have been underlined in a pilot study on 22 breast cancer biopsies using the mAh pAb 1801 for p53 determination. Formalin was compared with Bouins fixative, with no difference in the results obtained (37,60). However, the fixation time was of importance. Six h formalin fixation was compared with 24 h fixation. Fifteen of 22 samples fixed for 24 h completely lost their immunoreactivity. Interestingly, microwave treatment retrieved the p53 antigen in all cases but one (60). 

It is essential to use a formaldehyde-based fixative. The formaldehyde-based Bouin fixative see Note 16) gives the same results. The coupling reaction of... 

Bouin s solution is one of the traditional ways to harden cell pellet. Some cytologists believe it provides the best cellular details, especially nuclear features in cell blocks.28 The major steps are (1) After centrifugation, fix the cell pellet with Bouin s solution. (2) After 2h, discard the solution. (3) Remove the hardened cell pellet from the tube, wrap it with lens paper, and transfer it into a cassette for further processing. We have been using this method for many years. In our experience, most of the time, ICC results are consistent with IHC from the surgical specimen. The biggest drawback of this method is the toxicity of Bouin s fixative which creates biohazard and safety issues for the laboratory. We also found cell blocks gave poor fluorescence in situ hybridization (FISH) results after Bouin s fixation. 

Add approx five times the volume of the appropriate fixative (see Chapter 8) to the sample in a small wide-mouth vial and incubate for 4-24 h depending on the optimum for the fixative selected (formalin, 6-12 h Bouin s, 4 h ethanol/acid, 24 h) (see Note 3). 

Bouin s fixative has been used traditionally for the fixation of fetuses prior to fixed visceral examination. However, safety concerns over the use of picric acid have resulted in the use of alternatives such as Harrison s fixative (18). 

This chapter descrihes the method used for serial sectioning and soft tissue examination of the Bouin s fixed fetus (mainly whole rat and or mouse fetuses or rabbit fetal heads) for the assessment of developmental and structural abnormalities. Fetuses are examined externally, together with the internal structures of the head as well as the thoracic and abdominal organs. 

As soon as possible after removal from the uterus, the fetuses are euthanized and fixed in Bouin s solution. Fixation takes 7-14 days (see Note 2). Bouin s is the preferred fixative as it is sufficiently acidic to act as a decalcifying agent, which facilitates sectioning through bone, particularly the skull. Specimens may require hardening, by immersion in IMS for at least 24 hours to aid in sectioning. 

Loss of information (the skull bones of fetuses fixed in Bouin s fluid cannot be evaluated as this fixative decalcifies bone)... 

Formaldehyde, a monoaldehyde, is preferred over glutaraldehyde (a dialdehyde) because the latter introduces mostly irreversible protein crosslinks, masking the epitopes. Formaldehyde penetrates rapidly into the tissue but crosslinks proteins slowly. Most of these crosslinks are reversible, and therefore masked epitopes can be easily unmasked by treatments such as heating. However, for better preservation of cell morphology, a mixture of formaldehyde (4%) and glutaraldehyde (0.01-0.1%) can be tried. Bouin s fixative with its acidic pH should not be used. 

This need may arise when a limited number of slides is available. For this type of study, heat-induced antigen retrieval should be carried out only once. One such treatment lasts for many months. Repeated use of antigen retrieval tends to cause background staining. Such an approach has been successfully used for staining epithelial membrane antigens and cytokeratin in the tonsil tissue fixed with Bouin s fixative (Roche et al 2000). 

For the HercepTest to be valid, it must be performed exactly according to the manufacturer s directions. The tissue must be fixed in 10% neutral buffered formalin or Bouin s solution. Sections of paraffin-embedded tissues should be cut 3-A xm thick and processed in a standard tissue processor. Epitope retrieval should be carried out in a water bath instead of in a microwave oven. Water is used because it provides more uniform and... 

Formaldehyde, Fekete s acid-alcohol-formalin, Bouin s solution or other tissue fixative... 

Enumeration of gross metastases following fixation in Bouin s fixative or injection of Indian ink to increase contrast between the tumor and the parenchyma. 

Fix the spinal cord in the intact vertebral column using Bouin s fixative, either by immersion after dissection or by transcardial perfusion prior to dissection. The bone of the vertebrae will eventually decalcify in Bouin s (requiring 2-4 weeks), or after 24 h, the sample can be transferred to decalcifying solution overnight. 

Muscles can be fixed by perfusion or immersion in Bouin s fixative, dehydrated, and embedded in paraffin (record body weights and muscle weights following dissection, prior to processing). 

Tissue preservation All fetuses retained in Bouin s fixative or... 

Tissue preservation Malformations are developmental deviations that (1) are gross structural changes, (2) are incompatible with life, or (3) may affect the quality of life. Variations are structural deviations that are thought to have no effect on body conformity or the well-being of the animal. All fetuses are retained in Bouin s fixative or glycerin... 

Fig. 8.4. Visualization of lung metastases following staining with Bouin s fixative (lighter colored nodules on the surface, Panel A, tw o lungs to the right of the normal lung) or following injection of India ink solution into the trachea followed by destaining with Fekete s solution (Panel B, depicting white nodules against the...

In contrast to primates, which abort dead embryos, dead rodent embryos are resorbed and the implantation site is recorded as a resorption site. The numbers of living and dead fetuses, and the number of resorption sites, are counted, and fetal weight, sex, and external malformations are recorded. Fetuses can either be inspected fresh in evaluation of internal soft tissues (known as the Staples technique) or can be placed in a fixative (usually Bouin s solution) and sectioned at a later time (Wilson technique). Other fetuses are fixed in ethanol, cleared in potassium hydroxide, and the cartilage and bone are stained with Alcian Blue and Alizarin Red, respectively. The US FDA recommends that one-third of the rodent fetuses be subjected to visceral examination and that two-thirds be studied for abnormalities of cartilage and bone. The US Environmental Protection Agency (EPA) recommends that one-third to one-half of each litter be examined for skeletal anomalies. For rabbits, all fetuses are to be examined for both visceral and skeletal malformations. 

Hemmer and coworkers64 compared the influence of three different fixatives on immunoreactivity of P-gp antigen in tissue sections and reported that fixation conditions can strongly influence the staining results. Generally, Bouin s fixative was found to be inferior to Dietrich s or Lillie s fixative. Formalin was not considered in... 

Bouin s Fixative (see Note 7) Mix together 300 mL saturated picric acid (see Note 8), 100 mL commercial formalin (see Note 9) and 20 mL glacial acetic acid. 

Bouin s Fixative (see Note 18) 1. Fix tissue by immersion for 1-12 h depending on sample size (see Notes 19 and 20). 2. Wash in 70% alcohol to precipitate soluble picrates. 3. Wash in water. 4. Dehydrate and embed tissue in paraffin. 5. Cut sections may be treated with 5% aqueous sodium thiosulfate followed by washing in water to remove any residual picric... 

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