Microsporidia - IFT

– Filter through 50 µm (best) or 100 µm filter.

– Centrifuge at 700g for 15 minutes, discard the supernatant and resuspend the pellet (dilution 3 times) with PBS.

– Put 2 µl of the fecal sample suspension to be tested on well slides and dry for one hour. Count 2 wells per sample: 1 for each monoclonal antibody.

– Fix the slides with methanol, let dry 5 minutes.

– Add 20 µl of either of the monoclonal antibodies on the wells and incubate for 30 min at room temperature in a humid atmosphere.

– Wash 3 times with a drop of PBS, aspirating the PBS on the side of the wells without touching the sample.

– Add 20 µl of conjugate to each well. Incubate for 30 min at room temperature in a humid atmosphere, in the dark.

– Aspirate the conjugate, wash with a drop of PBS as specified above. Dip the slides in 3 changes of PBS.

– Drain the buffer from the slides. Dry the slides underneath and carefully around the samples, without touching the sample.

– Add 2 drops of anti-fading fluorescence mounting medium, place a coverslip on the wells, avoiding the formation of air bubbles.

– Reading is made with a fluorescence microscope, equiped with the appropriate fluoresceine filter (488 nm) and an immersion objective (x 1000).

2-8°C

Monoclonal antibody anti- E. bieneusi, monoclonal antibody anti- E. intestinalis, conjugate