Hi!

I am pursuing my research following the next protocol for inmuno assay in PCL electrospun meshes:

- Fix the samples with 4 % PFA for 1h in RT

- Clean 3X with DPBS for 3min each cleaning

- Permeabilized with DPBS + 2 % BSA + 0.3 % Triton X-100 for 1h in RT

- RInse 3X DPBS for 3min

- Add primary antibody in DPBS + 0.5% BSA through the mesh. I spend quite a lot to cover the whole sample.

- Incubate overnight 4ºC

- Cleaning 3X DPBS 3min each

- Add secondary antibody in DPBS + 0.5% BSA (1h in RT)

- Cleanning process

- Add DAPI for two minutes

- Cleaning process

- Place the electrospun mesh in a cover for further visualization in fluorescence microscopy or confocal.

The images I have obtained have room for improvement. I see a lot of background using Phalloidin or DAPI, the fibers are stained and the whole images gets a lot of signal.

¿Does anyone use fluorogel for mounting the samples?

¿Any sugestion in placing the antibodies, their concentration, cleaning processes or any tip to improve the quality of the images?

Thank you very much!!