Immunofluorescence

Immunofluorescence protocol

·      Collect tissue samples in a 2ml Eppendorf tube containing cold phosphate buffered saline (PBS).

·      Fix tissue as soon as possible for 10–20 min in 4% paraformaldehyde in PBS

·      Remove fixative solution and rinse with 3 washes of cold PBS

·      Block overnight in PBST (PBS + 0.1% Triton-X) + 5% donkey serum

o   Perform all incubations and wash steps on a rocking platform at 4 degrees Celsius (4C)

·      Remove block, add primary antibody diluted in 500ml PBST + 5% donkey serum, incubate ~24 hours at 4C

o   All primary antibodies used in this study were used at a dilution of 1:300

o   All secondary antibodies were used at a dilution of 1:1000

·      Remove primary antibody, rinse 3 x with PBST. Wash ~24 hours in 1ml PBST at 4C.

·      Add 1ml secondary diluted in PBST, incubate ~24 hours at 4C.

·      Remove secondary, rinse 3x with PBST, Wash ~24 hours in PBST at 4C.

·      Ready to mount/clear/image

Notes:

Staining outcomes are influenced by the amount of starting tissue, the quality of the tissue preservation, incubation times and temperate, and the batch of specific antibodies used. The timing of antibody incubations can be adjusted depending on the thickness of tissue and whether deeper tissue labelling is required. Allowing sufficient time for wash steps is important to improve signal to noise ratio for imaging. Antibody dilutions given above indicate a starting point that works for most antibodies. Optimisation of concentration can be performed as required.

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