Protocolo imunofluorescência

Double immunofluorescence – simultaneous protocol

1. Paraffin-embedded sections

1.1. Desparaffinize sections in xylene 3x5min (15 min).

1.2. Hydrate with 100% ethanol 3x3min (10 min).

1.3. Hydrate with 90% - 70% ethanol 1min (5 min).

1.4. Rinse in distilled water and then follow procedure for fixation and antigen retrieval as required (please see IHC protocol for formalin-fixed paraffin-embedded tissue sections for futher details).

1.5. Banho maria citrato 10mM pH6,0 ↑ 20min ↓ 20min.

2. Permeabilization

2.1. If the target protein is expressed intracellularly, it is very importante to permeabilize the cells. Note: acetone fixed samples do not require permeabilization.

2.2. Incubate the samples for 10 min with PBS containing 0,25% Triton X-100 (or 100 µM digitonin or 0.5% saponin). Triton X-100 is the most popular detergent for improving the penetration of the antibody. However, it is not appropriate for the use of membrane-associated antigens since it destroys membranes.

2.3. Wash cells in PBS three times for 5 min (15 min).

3. Blocking and simultaneous incubation

3.1. Incubate cells with 2% BSA (20mg – 1ml) in PBST for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum from the species that the secondary antibody was raised in).

3.2. Incubate cells in the mixture of two primary antibodies (e.g. rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) in 1% BSA in PBST in a humidified chamber for overnight at 4ºC.

3.3. Decant the mixture solution and wash the cells three times in PBS, 5 min each wash.

3.4. Incubate cells with the mixture of two secondary antibodies which are raised in different species (with two different fluorchromes, i.e. Texas Red-conjugated against rabbit and FITC-conjugated against mouse) in 1% BSA for 1h at room temperature in dark.

3.5. Decant the mixture of the secondary antibody solution and wash three times with PBS for 5 min each in dark.