Weak staining

Low concentration of primary antibody or short incubation time. 

Dilution of reagents due to inadequate removal of wash buffer between steps. 

Counterstain or mounting media are incompatible, which may dissolve the chromogen reaction product. 

Low target expression level or target damage including chemical modification due to fixation, over-fixation or paraffin embedding. 

Excessive incubation with protein blocking reagents (normal serum or commercial reagents). 

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Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed smears, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported otherwise the results will be of doubtful v alue. 

A second staining with Coomassie Brilliant Blue following the silver stain perhaps produces bands of proteins not or weak stain-able by silver, because silver staining is, on the one hand, more sensitive than other procedures, but on the other hand, also not universal. 

Note that the use of higher concentrations of monoclonal antibodies does not improve weak staining in immunohistochemistry because of paucity of or masked antigens. 

FIGURE 6.7. Immunostained PCNA in the hyperplastic human palatine tonsil, using mouse monoclonal antibody PC10 (diluted 1 10). (A) No heat pretreatment nuclei of the germinal center cells are weakly stained. (B) In contrast, the cells in the same area are strongly stained after simple heating in a hot water bath for 2 hr at 90°C. Reproduced, with permission, from Kawai et al. (1994). Copyright 1994 Blackwell Science Asia. 

Although the two methods mentioned above are viable options, they should not be applied independently of each other. A tested, standardized, and well-controlled immunohistochemical assay should serve as the first test. It is simple to carry out and quite reliable for the two extreme ends of the staining spectrum (9 and 3+) (Hendricks, 2000). The 2+ cases (weak staining in at least 10% of neoplastic cells) can in turn be subjected to FISH analysis to confirm the presence of an altered HER-2Ineu gene. 

An example of the way in which cell line controls can be used is illustrated by Dako s HercepTest kit, which contains three cell line controls a negative, a 1+ (weak staining) and a 3+ (strong staining). All are designed to be placed on the same microscope slide. 

Zebrin I compartmentalization in Saimiri sciurus was studied by Leclerc et al. (1990). Both in the vermis and the hemispheres clusters of Zebrin I-immunoreactive Purkinje cells were separated by weakly stained Purkinje cell somata or unstained cells. Zebrin-negative bands, therefore, are less distinct than in rodents. P1+, P2+ and P3+ bands are continuous from lobule to lobule and become narrower in the anterior lobe. P4+-P7+ bands were tentatively identified in the hemisphere, but not analysed in detail. A complementary histochemical zonation was detected for cytochrome oxidase, that was present in patches in the granular layer corresponding to the P- bands both in squirrel monkey and rat cerebellum. It is obvious from a comparison of the illustrations from the paper of Dore et al. (1990), showing the distribution of Zebrin I immunoreactivity in Purkinje cells and their axons and the zonation of AChE in monkey cerebellum, that the P2+ immunoreactivity in the anterior vermis corresponds to the X zone, and P2-... 

Fig. 156. Effects of promotor gene truncation on L7-lacZ banding pattern in mice. Cerebella were dissected free from the rest of the brain and stained in whole mount. Cerebella are viewed from posterior (POST) and anterior (ANT). Cerebella were taken from postnatal day 11 animals carrying 4 kb (top row), 500 bp (middle row) and 350 bp (bottom row) promoter constructs. The patterns are very similar to the Zebrin pattern in mouse cerebellum (compare Fig.139). Expression of L7 is absent or low in Pl-i-, P3+, P5+ (indicated with PIN in bottom panels) and P7-i- in the 500 and 350 bp constructs. In P4-i- there is a strong expression of L7 in the 500 bp construct, and a weak expression in the 350 bp construct. Reversed levels of expression are observed for the region of P4b-t- and P5a-t. In the 500 bp construct they are weakly stained (but the P4b-t and P5a + bands are visible as separate strips), in the 350 bp construct there is a strong expression of L7 over the entire area of these bands indicated with FN in lower pannel). Oberdick et al. (1993), interpretations by the authors of this chapter.

Under-fixation Background block Focal or weak staining... 

Technical Problems and Solutions Absence of Staining or Weak Staining ... 

WEAK STAINING OF SPECIMEN WITH APPROPRIATE STAINING OF POSITIVE CONTROL... 

Improper fixation or processing, or both, of the test tissue may also cause weak staining of the specimen, whereas the control stains positively. All the causes... 

FIGURE 8.19 Metastatic carcinoma In a lung vascular space (A). The tumor cells were diffusely and strongly positive for thrombomodulin (B) and showed only focal weak staining with uroplakin III (not shown), confirming urothelial differentiation of the tumor cells. 

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