Troubleshooting

Experimental pitfalls

(CARD-FISH not yet included...)

 

Chemicals

 

  • Always use fresh formamide - take an aliquot from the stock and use it up within a week. Formamide is quite delicate and can degrade leading to unspecific FISH signals. Also make sure that it is deoinized.

 

No cells stained ...

 

  • (i) check probe in photometer wether oligonucleotide and label are present in equimolar amounts, (ii) check probe on a polyacrylamide gel, only a single band representing intact probe should be visible
  • cells are overfixed resulting in PFA cross-links cell wall components and therefore in cells that are completely impermeable to probes (this may occur when cells stored for a long period in PFA) - try an increasing enzymatic digestion of the cells with e.g. Lysozyme, Proteinase K, Achromopeptidase, or incubate in 0.1 M HCl
  • no SDS in hybridization buffer - SDS denatures the native ribosome structure by removing the ribosomal proteins leading to an increased accessibility of the target sites

 

Unspecific staining of cells ...

 

  • (i) check probe in photometer wether oligonucleotide and label are present in equimolar amounts, (ii) check probe on a polyacrylamide gel, only a single band representing intact probe should be visible
  • recheck the sequence of the probe ordered
  • use a pencil (not a marker) to label your slides and filters - marker dyes are often fluorescent
  • prepare a fresh formamide stock

 

Low FISH signals ...

 

  • probe target site is inaccessible - try to use helper probes
  • for probes labeled with fluorescein derivatives, check if pH of mountant/resuspension buffer exceeds pH 9.0 (fluorescence maximum of fluorescein is pH >9.0)
  • use an alternative mounting reagent
  • try to use CARD-FISH

 

No cells visible ...

 

  • fixation was too short and cells are lysed - try to fix longer or fix the remaining sample again

Quenching

Even though there have been reports on the influence of quenching on the signal of the probe conferred fluorescence, for in situ hybridization this effect is negligible compared to the variation in signal among parallels (in the range of ~20%) (Behrens et al., 2003).

Probe Design

  • A problem that should be considered during the design of FISH probes is target site accessibility. The higher order structure of the ribosome may hinder the binding of the probe to its target site. The 16S/18S rRNA in situ accessibility for oligonucleotide probes has been studied for two members of the domain Bacteria (Escherichia coli, Rhodopirellula baltica (formerly Pirellula sp. strain 1)), one Eukaryote (Saccharomyces cerevisiae), and one Archaeon (Metallosphaera sedula) (Fuchs et al., 1998; Behrens et al., 2003). In 2001, a complete accessibility map for the 23S rRNA of E. coli has also been published (Fuchs et al., 2001). These color-coded maps clearly demonstrate dramatic differences in the binding of different fully complementary probes of approximately identical length to one batch of fixed target cells. Even though the secondary structure of the ribosome is highly conserved and a consensus map could be developed from the 16S/18S rRNA accessibility studies (Behrens et al., 2003), each probe should be checked on their respective target group of organisms to ensure high probe signals.
  • Yilmaz and coworkers showed that a longer hybridization time may compensate low binding efficiencies. There is an affinity and kinetic effect for at least some of the tested probes with low target site accessibility (Yilmaz et al., 2004). By carefully changing the probe length and hybridization times they were able to virtually open every target site for oligonucleotide probes.
  • Alternatively, inaccessible target regions based on stable secondary structures can be made accessible using unlabeled oligonucleotides, called helpers (Fuchs et al., 2000). These helpers bind adjacent to the diagnostic probe and thereby opening the the target region for the probe. Helpers should be a few nucleotides longer than the diagnostic probe, i.e., if the probe is an 18-mer the helper should be a 21-mer to ensure a tight binding beyond the melting point of the diagnostic probe.