GeneFISH – combined detection of genes and rRNA in microorganisms

B3: Antibody binding

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

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 to geneFISH            

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

B3: Antibody binding

            Antibodies are prone to unspecific binding. To minimize it, blocking reagents have to be used. The blocking reagents and the antibody concentration necessary for reduction of background can be sample dependent. When stronger blocking is required, other blocking reagents can be used, either alone, or in combination (e.g. preimmune serum, bovine serum albumin and detergents).

Procedure

• Transfer filter in 50 ml antibody buffer and incubate with slow shaking (20 rpm) for 45 min
• Transfer filters in antibody solution (antibody buffer + antibody) and incubate for 1.5 h, RT, with slow shaking
• quick wash in antibody buffer
• 3x10 min wash in antibody buffer

Table 6: Antibody buffer

Antibody solution: antibody buffer + 1:500 dil of Anti Dig POD Fab Fragments (Roche), with a final concentration of 0.3 U/µl.

 

 

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

            In this step, the HRPs conjugated to the antibodies will catalyze the deposition of fluorescently labeled tyramides on the cellular proteins. For preparation of the tyramides see (Pernthaler and Pernthaler, 2005). The preferred fluorochrome in this step is Alexa594, as it gives a very strong fluorescence. After the tyramides deposition, an HCl treatment has been introduced, to inactivate the HRPs and avoid false positive signals during the rRNA detection step.

Procedure

• Place droplets of amplification solution (90 µl each) in PDs and add filters face down in the droplets
• incubate at 37°C for 45 min
• 10 min wash in 50 ml 1xPBS, at RT
• 5 min wash in miliQW
• 10 min in 0.05 M HCl (50 ml miliQW + 250 µl 37% HCl)
• 2 x 10 min wash in 50 ml 1x PBS, 46°C, slow shaking
• Quick miliQW
• Quick 96% ethanol
• Air dry
• Store filters at -20°C

100x H₂O₂: 200 µl 1x PBS + 1 µl 30% H₂O₂

Amplification mix

• 1 ml amplification buffer II
• 10 µl 100x H₂O₂
• 2 µl Alexa594-Tyramide

Preparation of 40 ml amplification buffer II for the gene CARD step

In a 50 ml Falcon tube, add the following (with exception of the blocking reagent):

• Shake to dissolve the dextran sulfate and incubate at 48°C (water bath) until dextran sulfate is dissolved
• Add 400 µl Blocking reagent (final concentration 0.1%)
• Vortex, spin
• Filter through 0.22 µm filter, Millipore
• Store @ +4°C

References:

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.