GeneFISH – combined detection of genes and rRNA in microorganisms

C. rRNA detection

 

 

 

 

When you are using this protocol, please cite:

Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ. Microbiol. 12: 3057-3073.

 

 

Introduction           

A. Sample preparation

A1. Sample fixation

A2. Sample immobilization

A3. Embeding in agaroze

A4. Permeabilization

A5. Inactivation of endogeous peroxidases

B. gene detection

B1. gene probes

B1.1. probe design

B1.2 probe synthesis

B1.3 determination of hybridization conditions for hybridization and washing

B2. gene hybridization

B3. antibody binding

B4. gene CARD and inactivation of HRPs from the gene detection step

C. rRNA detection

C1. rRNA hybridization

C2. rRNA CARD

D. Mounting for microscopy and counterstaining

E. Microscopy

F. List of materials

C. rRNA detection

            The rRNA detection step is performed as in the regular CARD FISH protocol (Pernthaler et al., 2002), with probes specific for the clades of interest. For preparation of the tyramides see (Pernthaler and Pernthaler, 2005).

C1: rRNA hybridization

Procedure

• ~ 1.2 ml 35% FA hybridization buffer + 4 µl EubI-III probe, mix.
• Place 50 µl droplets of hybridization mix in PDs, add filters face down
• Place PDs in hybridization chamber
• 3 h hybridization at 46°C
• Prewarm washing buffer at 48°C
• Quickly rinse filters in washing buffer
• Transfer filters in new washing buffer, wash for 15 min at 48°C

Table 2: Washing buffer for 48°C, 35%FA HB

C2: rRNA CARD

            In this step, the HRPs from the oligonucleotide rRNA probes will deposit fluorescently labeled tyramide on the cellular proteins. The preferred fluorochrome for this step is Alexa 488, as it can be used in double hybridizations with Alexa 954.

Procedure

• incubate filters in 1x PBS, ~ 20 min at RT
• transfer filters in amplification mix (1x PBS containing 1x H₂O₂ and 1:3000 dilution of Alexa 488 tyramide)
• incubate for 10 min at 37°C
• quick wash in 1x PBS from the previous step
• 10 min wash in new 1x PBS
• 1 min in miliQW
• 1 min in 96% ethanol
• Air dry

Amplification mix:

4 ml 1x PBS

40 µl 100x H₂O₂ (200 µl 1x PBS + 1 µl 30% H₂O₂)

1 µl Alexa488-Tyramide

 

References

Pernthaler, A., Pernthaler, J., and Amann, R. (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68: 3094–3101.

Pernthaler, A., and Pernthaler, J. (2005) Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes. Methods Enzymol 397: 351-371.