Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

• Publication year

  1994

• Venue

  Journal of Cell Biology

• Publication date

  1994-08-02

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1083/JCB.126.4.901PMID 7519623PMCID 2120127
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Fluorescence photooxidation can also localize specific nucleic acid sequences by in situ hybridization using biotinylated probes followed by an eosin-streptavidin conjugate.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• Eosin's smaller size than conventional markers improves penetration of labeling reagents relative to gold- or enzyme-based procedures.

  Confidence 0.90

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• Correlative immunolocalization on the same sample is enabled across fluorescence, transmitted light, and electron microscopy.

  Confidence 0.94

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• Eosin increases the sensitivity of fluorescence photooxidation for converting diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer compared with conventional fluorescent compounds.

  Confidence 0.97

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review

• biotinylated probes

  probe, molecular reagent

  Biotin-tagged nucleic acid probes used to detect target sequences in hybridization assays.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• correlative immunolocalization

  imaging workflow, method

  A same-sample localization workflow that combines fluorescence, transmitted light, and electron microscopy for labeled proteins.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• diaminobenzidine tetrahydrochloride

  chemical, chromogen

  The chromogen oxidized into an insoluble osmiophilic polymer during photooxidation.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• eosin

  fluorescent dye, marker

  A fluorescent dye used as the marker to drive photooxidation in this protocol.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• eosin-streptavidin conjugate

  conjugate, labeling reagent

  A streptavidin-linked eosin label used after biotinylated probes in hybridization detection.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• fluorescence photooxidation

  method

  A fluorescence-driven oxidation method that converts diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer for microscopic visualization.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• high voltage electron microscopy

  microscopy technique

  An electron microscopy modality used for three-dimensional imaging of heavily labeled samples.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review
• in situ hybridization

  molecular detection method

  A nucleic acid detection method applied here to localize specific sequences in cells and tissues.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAK (4715169a40) reviewjihoonc (k5vuy3tzcm) review

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