Protocols

Here, we provide a number of general FISH protocols which should serve as a basis for a successful hybridization.

 

Note, that these protocols only represent a guideline. There are many parameters which have to be optimized for your specific sample, you are working on. It may well be, that for every new sample such an omptimization is required. However, we will point out for which steps it is worthwhile to play with the different experimental conditions.

General overview

FISH of whole cells always consists of four parts:

 

  1. Fixation of the sample containing the target cells. Fixation stabilizes macromolecules and cytoskeletal structures thus preventing lysis of the cells during hybridization. At the same time fixation permeabilize the cell walls for the fluorescently-labeled oligonucleotide probe molecules.
  2. The fixed cells are incubated (hybridized) in a buffer containing the labeled probe at a specified temperature which favours the specific binding of the probe to the target. Ideally, only those probe/rRNA pairs will form which have no mismatches in the hybrid. Consequently, only target cells that contain the full signature sequence on their rRNA will be stained.
  3. The subsequent washing step will remove all unbound probe molecules.
  4. Finally, the hybridized cells are enumerated by epifluorescence microscopy or by flow cytometry.

Protocols for download

(LAST UPDATE October 25, 2010)

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