Staining artifactual

Sections should never be allowed to dry during or after the HIER procedure, as this may result in artifactual staining. 

If the tissues have not been appropriately snap frozen, ice crystals will be present in the tissue, identifiable as slits or fracture lines through the tissue. Artifactual staining may occur along such fracture lines. Therefore, ice crystal damage should be avoided. The tissues should always be snap frozen. If freezing is performed slowly over dry ice, ice crystals will accumulate in the tissue. 

NOTE Use powder-free gloves when handling all materials for this procedure and handle membranes with forceps at the edges to avoid potential artifactual staining. 

TABLE 1.11 Technical Problems and Solutions Artifactual Staining ... 

Presumably because of the relatively high content of phenolic amino acids, products based on milk are strongly autofluorescent when viewed after either 360 nm or 490 nm wavelength excitation. Methods for fluorescence reduction such as prior staining with Toluidine Blue O or Evan s Blue can be employed, especially on sectioned material where artifactual collapse of the emulsion which is caused by the charged nature of the dye, is not an issue [22],... 

The staining is carried out with 100 pi of 4% uranyl acetate in 50% ethanol for 1 min in a microwave oven set at a power level of 125,6W, followed by rinsing with 500 ml of distilled water. This is followed by staining for 1 min with 100 pi of triple lead citrate (Sato et al., 1988) and then rinsing with 500 ml of distilled water. This lead citrate staining solution avoids the production of artifactual lead carbonate precipitates. 

A variety of enzymes can be used for incorporation of the label. Klenow fragment of DNA polymerase I is preferable to the DNA polymerase I holoenzyme because it has the identical polymerase activity, but lacks the exonuclease activity that could cause artifactual labeling (43). Alternatively one can use TdT (33), which adds on long tails of nucleotides to the 3 hydroxyl ends of DNA without the need for a template strand (43). It is extremely important to use the correct concentration of enzyme, as increased amounts will lead to nonspecific staining of morphologically normal nuclei (34,35). Obviously, insufficient enzyme will lead to a reduction in the staining of apoptotic nuclei. 

The pitfalls of interpretation of lymphatic channels in paraffin-embedded breast tissue are well known. Retraction artifacts, ducts with misplaced epithelium, and artifactual displacement of cells commonly complicate the interpretation of biopsy samples. A recently available antibody, D2-40, shows high sensitivity and specificity for normal lymphatic channels in a variety of tissues. 493 D2-40 stains the lymphatic endothelium crisply and intensely but does not stain the normal vascular endothelium (Fig. 19.30).It is highly sensitive and specific in identifying lymphatic space invasion. 

Artifactual bands with molecular weights ranging from 50,000 to 68,000 are often observed in silver-stained gels. Evidence has been presented indicating that these bands are due to contamination by keratin-type proteins (Ochs, 1983). These bands may be more prominent in the presence of certain reducing agents, such as mercaptoethanol. 

A completely random pattern of fuzzy stain patches and streaks, such as might result from artifactual disorder of membrane components, would also delineate particles of random size and shape the size range, limited approximately by that of the membrane width, and the size distribution would necessarily correspond to those observed. That the upper limit for mitochondrial proteins (assumed to be spherical) be approximately 100 A is all that is required to ensure the type of correlation observed. 

Objects examined in experiments previously discussed were not particularly deformable structural units. What happens with material of lower cohesiveness is illustrated by work on PTA staining of lipid bilayer systems. Discussing the micrograph in Plate II of their article, Bangham and Home (1964) attributed the tilting of dimer units of lecithin (44.2 X 13 A) in lipid bilayers to ionic interaction with PTA located in adjacent rows. In the present author s opinion, their explanation is all the more probable that one dimension in PTA and dimer phospholipids is approximately the same, 13 A. Hence artifactual tilting of dimer units may be induced by some specific pattern of adjacent PTA molecules. 

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