Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

Baschong, Werner; Suetterlin, Rosmarie; Laeng, R. Hubert

doi:10.1177/002215540104901210

Cited by 265 publications

(240 citation statements)

References 14 publications

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“…After extensive washing with 0.1 M KPBS, ephrinA5 expression was visualized with neutravidin™ fluorescein conjugate (Molecular Probes, Eugene, USA), diluted at 1/200 in blocking solution, for 45 min at RT. Following several washes with 0.1 M KPBS, slides were covered with mowiol anti-fade mounting medium [63,64]. …”

Section: Methodsmentioning

confidence: 99%

EphrinA5 protein distribution in the developing mouse brain

Deschamps¹,

Morel²,

Janet³

et al. 2010

BMC Neurosci

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Background EphrinA5 is one of the best-studied members of the Eph-ephrin family of guidance molecules, known to be involved in brain developmental processes. Using in situ hybridization, ephrinA5 mRNA expression has been detected in the retinotectal, the thalamocortical, and the olfactory systems; however, no study focused on the distribution of the protein. Considering that this membrane-anchored molecule may act far from the neuron soma expressing the transcript, it is of a crucial interest to localize ephrinA5 protein to better understand its function. Results Using immunohistochemistry, we found that ephrinA5 protein is highly expressed in the developing mouse brain from E12.5 to E16.5. The olfactory bulb, the cortex, the striatum, the thalamus, and the colliculi showed high intensity of labelling, suggesting its implication in topographic mapping of olfactory, retinocollicular, thalamocortical, corticothalamic and mesostriatal systems. In the olfactory nerve, we found an early ephrinA5 protein expression at E12.5 suggesting its implication in the guidance of primary olfactory neurons into the olfactory bulb. In the thalamus, we detected a dynamic graduated protein expression, suggesting its role in the corticothalamic patterning, whereas ephrinA5 protein expression in the target region of mesencephalic dopaminergic neurones indicated its involvement in the mesostriatal topographic mapping. Following E16.5, the signal faded gradually and was barely detectable at P0, suggesting a main role for ephrinA5 in primary molecular events in topographic map formation. Conclusion Our work shows that ephrinA5 protein is expressed in restrictive regions of the developing mouse brain. This expression pattern points out the potential sites of action of this molecule in the olfactory, retinotectal, thalamocortical, corticothalamic and mesostriatal systems, during development. This study is essential to better understand the role of ephrinA5 during developmental topographic mapping of connections and to further characterise the mechanisms involved in pathway restoration following cell transplantation in the damaged brain.

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Section: Methodsmentioning

confidence: 99%

EphrinA5 protein distribution in the developing mouse brain

Deschamps¹,

Morel²,

Janet³

et al. 2010

BMC Neurosci

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“…Secondary antibody incubation (AlexaFluor488, donkey anti-mouse and Alexafluor568, donkey anti-mouse, or AlexaFluor594, goat anti-rabbit; Invitrogen, Carlsbad, CA, USA) was performed for 30 min at room temperature, after which slides were post-treated for 30 min with 0.1% Sudan Black B (Fisher Scientific, Pittsburgh, PA, USA) dissolved in 70% ethanol as described. 34 Slides were cover-slipped with Prolong Gold Antifade Reagent containing DAPI (4 0 ,6-diamidino-2-phenylindole) nuclear counterstain (Invitrogen).…”

Section: Immunohistochemistry and Immunofluorescencementioning

confidence: 99%

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Flores-Figueroa

Varma

Montgomery

et al. 2012

Laboratory Investigation

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“…[6][7][8] However, AF is an obstacle to immunofluorescence (IF) analysis that can either mask or interfere with specific fluorescent signals, 9,10 especially those with weak IF labeling, including direct IF and multiple-color fluorescence staining. In addition to intrinsic fluorescence of kidney is AF that arises from the tissue-processing techniques, including fixation agents such as glutaraldehyde 11 and embedding material such as paraffin. 9,11 In general, AF interferes with routine specific fluorescent labeling by red, green, and blue fluorescent probes, making it very difficult to visualize or colocalize multiple proteins of interest.…”

mentioning

confidence: 99%

“…In addition to intrinsic fluorescence of kidney is AF that arises from the tissue-processing techniques, including fixation agents such as glutaraldehyde 11 and embedding material such as paraffin. 9,11 In general, AF interferes with routine specific fluorescent labeling by red, green, and blue fluorescent probes, making it very difficult to visualize or colocalize multiple proteins of interest.…”

mentioning

confidence: 99%

Sudan Black B Reduces Autofluorescence in Murine Renal Tissue

Sun¹,

Hong²,

Zheng³

et al. 2011

Archives of Pathology &Amp; Laboratory Medicine

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N Context.-Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material.Objective.-To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B.Design.-In this study, the sources and features of autofluorescence emitted from kidney tissue were examined. Broad autofluorescence was visualized in both frozen and paraffin kidney sections of normal mice and mice with Adriamycin-induced nephropathy using confocal laser scanning microscopy. Autofluorescence appeared in commonly used 49,6-diamidino-2-phenylindole, fluorescein isothiocyanate, and Texas Red channels but not in far-red channel, and emitted extensively from red cells, injured tubulointersitial cells, and protein casts in diseased kidney.To eliminate autofluorescence, Sudan black B was used on formaldehyde-fixed paraffin sections and frozen sections of mouse kidney. The effects of Sudan black B in various concentrations were tested on kidney tissue.Results.-The 0.1% Sudan black B effectively blocked autofluorescence from both paraffin and frozen sections without adversely affecting specific fluorescence signals. Interestingly, the solvent for Sudan black B, 70% ethanol, was also shown to reduce autofluorescence on frozen sections, but not on paraffin sections.Conclusions.-This study demonstrates a simple, efficient, and cost-effective method to reduce autofluorescence using Sudan black B, and also provides a comprehensive approach to identify and minimize autofluorescence of renal tissue.

show abstract

Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

Cited by 265 publications

References 14 publications

EphrinA5 protein distribution in the developing mouse brain

EphrinA5 protein distribution in the developing mouse brain

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Sudan Black B Reduces Autofluorescence in Murine Renal Tissue

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