Immersion fixation

In brief, the Feulgen staining procedure consists in (a) fixation of root tips in Camoy s solution I (ethanol and acetic acid, 3 1 v/v) (b) staining with Schiff s reagent and (c) two successive immersions in 95% ethanol and histolemon Erba baths. More details on the procedure can be found in Ferrara et al. (2001). 

Fixation by rapid freezing followed by either freeze substitution or cryosectioning can also overcome some of the problems of standard immersion fixation and resin embedding. These are more specialized techniques and will not be dealt with here. Discussion of the methods can be found in Polak and Varndell (6), Hayat (7), and Verkleij and Leunissen (8). 

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. 

In exhaustion dyeing, the dye, which is at least partially soluble in the dyebath, is transported to the fiber surface by motion of the dye liquor or the textile. It is then adsorbed on the fiber surface and diffuses into the fiber. Finally, depending on the dye-fiber interaction, it is fixed chemically or physically. The dye can be applied to the textile discontinuously from a dilute solution (exhaustion dyeing from a long liquor) or continuously by immersing the textile in a concentrated bath and squeezing-off excess liquor (padding), followed by separate steps for diffusion and fixation in the fiber. 

Stop Bath for Tropical Development Films should be immersed for three minutes in either Kodak SB-4 Tropical Hardener Bath or Kodak SB-5 Nonswelling Acid Rinse Bath after development and before fixation. Agitate the negative in the stop bath frequently. 

The fixing process involves a series of chemical reactions in which the silver bromide is converted into complex argentothiosulfates, which are then dissolved by contact with fresh fixer and finally washed out of the film or paper. Upon immersing an emulsion (film or paper unless otherwise noted) into fixer, the first reaction is the conversion of unused silver bromide into an insoluble but not very stable compound. This compound can be seen by looking at negatives (not prints) after only a few seconds in the fix. They will appear milky in appearance. If fixation is not continued and the compound not completely dissolved, the negatives will rapidly degenerate. 

While choice and validation of reagents are critical issues, in my opinion, by far the most important contributory factor to variable performance of IHC worldwide, as well as in individual laboratories, relates to the pre-analytic phase (6-10). Most important is the almost complete absence of consistency in tissue fixation, and other aspects of specimen handling. While formalin is by far the most widely employed fixative, there is no consistency in its preparation, whether freshly prepared or not, or in time for which tissues are exposed to the fixative. Indeed almost always the fixation time is unknown and undocumented. Similarly pre-fixation time (sometimes referred to as ischemic time — post resection, but prior to immersion in fixative) is not documented and is unknown in duration or impact. Similarly the other stages of processing, dehydration, impregnation,... 

Tissue Processing Skinned mouse knee joints are immersed in neutral buffered formaldehyde (3.7 %) directly after preparation. Fixation times are limited to one week to ensure tissue integrity. Tissue samples are rinsed overnight with tap water. Tissue samples that contain bone are decalcified (D-calciher normal, Shandon 1779) for 2 or 3 days. Decalcified samples or cartilage samples without bone are rinsed overnight with tap water. After dehydration with alcohol in stepwise increasing concentrations, tissue samples are embedded in paraffin. 

Frozen material or tissue-culture cells may be fixed by immersion for 10 min in 50% methanol/50% acetone at -20°C, followed by air drying. Alternatively, a 4% solution of paraformaldehyde can be prepared by dissolving 4 g of paraformaldehyde powder in 80 mL of water with gentle heating and the addition of 1 M NaOH until the powder dissolves. The solution is made up to 90 mL with distilled water, and 10 mL of a 10X PBS solution added. Fixation is for 10 min at 4°C, and slides are then rinsed in PBS. Tissues or cells which are to be used for TUNEL/ISEL should be fixed as quickly as possible, because delay causes significant artifacts. 

When peroxidase is used as the label, endogenous peroxidase should be inactivated (e.g., immersing slides, after fixation, in 1% H2O2 in methanol for 30 min, washing in methanol and air-drying. 

Mice are perfused with 20 ml PBS and 20 ml fixative (Table 11.3C) and tissues immersed in fixative at 4°C for 30 min. Fixation with formaldehyde is quenched in cold 0.15 M triethanolamine (pH 7.5) for 30 min and two 5 min washes in cold PBS. Tissues are dehydrated in graded ethanol and embedded in paraffin. After sectioning and adhering to a coated slide, paraffin is removed by dipping three times 5 min in xylene and twice 5 min in ethanol. 

For touch preps, the slide should be immersed in acetone as soon as possible, but the cells need a moment to adhere to the plane of the glass. Slowly dip the slide into the acetone as a violent action at this point could wash off some of the cells. As is the case with frozen sections, fixation is a matter of choice. In this instance the use of acetone is preferred because the cells are still whole and the membranes require disruption in order for the contents to be accessible for later analysis. However, an ethanol/acid fixative (95% ethanol, 5% glacial acetic acid) is perfectly acceptable if desired. For the other cytology specimens, experimentation within individual systems is necessary. There is no right or wrong way to make these preparations. There is a fine balance struck between the... 

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