Prevention of false positive binding during immunofluorescence of Staphylococcus aureus infected tissue biopsies

Tan, Neil; Tran, Hai B.; Roscioli, Eugene; Wormald, Peter John; Vreugde, Sarah

doi:10.1016/j.jim.2012.07.015

Cited by 8 publications

(1 citation statement)

References 15 publications

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“…One of the perennial problems researchers face when using this technique are false positives due to autofluorescence or non-specific antibody binding which, if not taken into account can lead to wrong conclusions (Baschong et al, 2001; True, 2008; Tan et al, 2012). Although no systematic studies of autofluorescence or non-specific staining of enamel were published, enamel researchers are generally aware of these issues and interpret immunofluorescence studies of amelogenesis with caution.…”

Section: Introductionmentioning

confidence: 99%

Optimizing Immunostaining of Enamel Matrix: Application of Sudan Black B and Minimization of False Positives from Normal Sera and IgGs

2017

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Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immunofluorescence studies and lead to false positives. Our goal was to assess degrees of non-specific binding under different conditions and try to optimize procedures for immunofluorescence studies of forming enamel. Firstly, we compared two methods for background fluorescence elimination, i.e., sodium borohydride and Sudan Black B treatments. The results demonstrated that Sudan Black B is far superior to sodium borohydride in reducing the background fluorescence in dental tissues. We also studied the extent of non-specific binding of normal sera and purified polyclonal immunoglobulins (IgG) from five mammalian species, guinea pig, rat, rabbit, goat, and sheep, over a broad range of dilutions. For all sera tested fluorescence signals increased exponentially from 1:1000 to 1:100. Interestingly, the non-specific binding of sera from rodent species was below that of positive control in the whole range of dilutions. In contrast, incubation with sera from 3 non-rodent species produced much higher signals which surpassed the positive control signal at 1:250~1:500 dilution range. Most of the IgGs didn't show significant non-specific binding within 0.25–5 μg/ml range, except rabbit IgG which demonstrated extremely high affinity to the enamel matrix even at concentrations as low as 1 μg/ml. Further, studies confirmed that Fab fragments of purified normal rabbit IgG, not conserved Fc fragments, were involved in the interactions. Our observations suggest this high affinity is associated with the antigen binding sites of rabbit IgG. We anticipate that our results will help enamel researchers to optimize and standardize their immunochemical procedures.

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Section: Introductionmentioning

confidence: 99%

Optimizing Immunostaining of Enamel Matrix: Application of Sudan Black B and Minimization of False Positives from Normal Sera and IgGs

2017

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Immunofluorescence Procedure for Developing Enamel Tissues

Yang

Beniash

2019

Methods in Molecular Biology

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Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy

et al. 2018

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Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining. This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride (NaBH) and Sudan black B (SBB) alone (p < 0.05). Under maximum effective concentration, semi-quantitative analysis revealed that inhibitory effect of SBB on AF was superior to that of NaBH (P < 0.01). When the two chemicals were combined, we observed that background can be reduced, and specific staining can be optimized at optimum concentration. Intensity of renal tissue was examined by confocal λ scanning, which showed that peaks were located at the range of approximately 480 - 590 nm and similar to those of flavin and lipofuscin. These results indicated that combined use of NaBH and SBB, when targeted at different sources of AF in renal tissue, is the most effective means of reducing background and preserving specificity of fluorescent labels. In addition, this method does not interfere with various steps of immunofluorescence experiments.

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Prevention of false positive binding during immunofluorescence of Staphylococcus aureus infected tissue biopsies

Cited by 8 publications

References 15 publications

Optimizing Immunostaining of Enamel Matrix: Application of Sudan Black B and Minimization of False Positives from Normal Sera and IgGs

Optimizing Immunostaining of Enamel Matrix: Application of Sudan Black B and Minimization of False Positives from Normal Sera and IgGs

Immunofluorescence Procedure for Developing Enamel Tissues

Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy

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