GeneFISH – combined detection of genes and rRNA in microorganisms

D. Mounting for microscopy and counterstaining

E. Microscopy

 

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

D: Mounting and counterstaining

 

            Embed filters in ProLong Gold or SlowFade Gold containing 2x DAPI. In the case of ProLongGold, the samples need to cure for 24 h at room temperature. For storage, place slides at -20°C.

 

E: Microscopy