|
Solution
|
Components
|
Volume
|
Final concentration
|
| Hybridization buffer: | 5-M NaCl | 360 µL | 900 mM |
|
|
1-M Tris/HCl (pH = 7.5) | 40 µL | 20 mM |
|
|
Formamide | Vol% depending on probe |
|
|
|
Distilled H2O | Add to 2 mL |
|
|
|
10% SDS | 2 µL | 0.1% |
| Washing buffer: | 5-M NaCl | Depending on %* | 900 mM |
|
|
Formamide in hybridization buffer (see above) | * |
|
|
|
1-M Tris/HCl (pH = 7.5) | 1 mL | 20 mM |
|
|
0.5-M EDTA | 500 µL | 5 mM |
|
|
Distilled H2O | Add to 50 mL |
|
|
|
10% SDS | 50 µL | 0.01% |
|
Hybridization procedure
|
Comments
|
||
| Resuspend sample and transfer 20-100 µL of aliquot to 500 µL of a 1:1 mix of PBS/ethanol in a 2-mL microfuge tube. | Optional dilution step. | ||
| Sonicate aliquot for 20-30 s at low intensity using 1-s sonication pulses. If required, the sonicated sample can be further diluted. | For fine-particle sediments, vortexing is fine. | ||
| Place GTTP filter (pore size = 0.2 µm) on glass filter surface (shiny side on top). Add 10-100 µL of fixed suspension to 5-10 mL of PBS buffer into the filter tower and filter this volume onto the membrane filter. |
|
||
| Air-dry filters can be stored in Petri dishes at -20°C until hybridization. | The procedure can be paused here if shipboard schedule demands it. | ||
| Prepare 2 mL of hybridization buffer in a microfuge tube (see above). |
|
||
| For the hybridization mixtures, add 2 µL of probe working solution (50-200 ng probe/µL) to 18 µL of hybridization buffer in a 0.5-mL microfuge tube. | Keep probe solutions dark and on ice. | ||
| Cut filter in quarters with razor blade and place the pieces on a glass slide, upside facing up. | Do not touch filter surface with fingers. Several filters can be placed on one slide and simultaneously hybridized with the same probe. Quarters may be marked with incisions if different probes will be applied. | ||
| Put a piece of blotting paper into a 50-mL polyethylene tube and soak it with the remaining hybridization buffer. | JOIDES Resolution toilet paper can serve as blotting paper. | ||
| Add hybridization mix on the samples, one 20-µL drop per filter section, and place the slide with filter sections into the polyethylene tube in a horizontal position. | Make sure the hybridization mix does not run off. | ||
| Incubate 1.5-2 hr at 46°C. | Do not exceed 3 hr. | ||
| Prepare 50 mL of washing buffer (see above) in a polyethylene tube and pre-warm at 48°C in a water bath. |
|
||
| Quickly transfer filter sections into preheated washing buffer and incubate for 15 min at 48°C. |
|
||
| Pour washing buffer with filter sections into a Petri dish. Pick filter sections and rinse them by placing them into a Petri dish with distilled H2O for several seconds, then let them air-dry on blotting paper. |
|
||
| For counterstaining, put filter sections on glass slides (side with bacterial cells facing up), cover with 50 µL DAPI solution (1 mg/L), and incubate for 3 min. Afterward, rinse filter sections briefly in distilled H2O, wash them for several seconds in 80% ethanol to remove unspecific DAPI staining, and air-dry. |
|
||
| Filters on glass slide are embedded in a 4:1 mix of Citifluor and Vecta Shield and are covered with a cover slide. The filter sections have to be completely dried before embedding; otherwise, a fraction of cells will detach during inspection. | It is also possible to use Citifluor or Vecta Shield without a coverslip. | ||
| Double-stained and air-dried preparations as well as filters mounted on slides can be stored in the dark at -20°C for several months without substantial loss of probe fluorescence. |
|
||
| Probe-conferred fluorescence fades much more rapidly than DAPI fluorescence in the microscopic image. For counting, it is therefore safer to first quantify specifically stained cells in green excitation and subsequently all cells from the same field in UV excitation (DAPI). |
|
||
Notes: FISH = fluorescence in situ hybridization. PBS = phosphate buffered saline, DAPI = 4´,6-diamindino-2-phenylindole, Tris = tris(hydroxymethyl)amino methane, SDS = sodium dodecyl sulfate, EDTA = disodium ethylenediaminetetraacetate, UV = ultraviolet. * = concentrations of NaCl in washing buffer (48°C) are given at different concentrations of formamide in hybridization buffer (46°C). The first number of each pair is the formamide concentration in the buffer (vol%) and the second number is the NaCl concentration in the washing buffer (mM): 1, 900; 5, 636; 10, 450; 15, 318; 20, 225; 25, 159; 30, 112; 35, 80; 40, 56; 45, 40; 50, 28; 55, 20; 60, 14; 65, 10; 70, 7; 75, 5; 80, 3.5.