- Excise intestine in 0.5cm sized pieces.
- Fix by immersion in 4% Paraformaldehyde in PBS, overnight at 4°C, slowly rocking. I would do this so that each piece of intestine is in a 2ml eppendorf tube.
- Slice tissue into 2mm X 2mm pieces.
- Rinse 3X 20min per rinse in PBS, to remove PFA
- Permeabilise in 1% Triton X-100 in PBS for 2hr, slowly rocking , room temp.
- Block 1hr in 1% BSA, 3% Normal Goat Serum, 0.2% TX-100 in PBS
- Stain overnight rocking slowly in the dark at 4°C in 1 ml of PBS with 0.1%BSA, 0.3% Normal Goat Serum, 0.2% TX-100 containing 5 micrograms/ml of DAPI and 0.1 Units of the fluorochrome coupled phalloidin (either Alexa 488 or Alexa 568)
- (Steve titrated down both DAPI and phalloidin concentrations in April of 10 – only need 5µg/ml of DAPI and 0.1U/ml of the fluorescent phalloidin – NB this is for confocal use instead of 2 photon as in Appleton et al.)
- Rinse 3 X 20min in PBS.
- Mount in 97%TDE (2,2’-thiodiethanol).
- Mounting is achieved by immersing tissue in increasingly concentrated dilutions of TDE. 2h at 10%, 2h at 25%, 2h at 50%, finally O/N at 97%.
- Place the tissue piece (inside of intestine facing down) onto a 35mm glass bottomed dish (tissue culture dish with a number 1.5 coverslip in the bottom) or a 30 or 40 mm diameter number 1.5 coverslip.
- Hold tissue in place by placing a 12 mm coverslip on the top of the tissue.
Appleton et al. Preparation of whoemount mouse intestine for high resolution three-dimensional imaging using two-photon microscopy. Journal of Microscopy (2009) vol. 234 (2) pp. 196-204
Staudt et al. 2, 2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc Res Tech (2007) vol. 70 (1) pp. 1-9