Day 1:

Preparing the slides/plates/dishes:

·         Pipette about 200µl Poly-L-Lysine to cover the area on the dish. Leave it for 15 minutes.

·         Remove and give a water wash.

·         Put them on 55-60°C oven for about 15 minutes to dry it.

Formaldehyde fixation

·         Dissect about an average 5 pairs of tubules per plate.

·         Just before transferring the tubules on the dish, add about 100µl 1xPBS on the plate.

·         Transfer the tubules as quickly as possible and try to stick them on the dish with glass rod or forceps without breaking the tubules.

·         Remove PBS with a pipette and add (fix) with 4% Paraformaldehyde in PBS. (20 minutes)

·         On the shaker, give 3x10 min. washes with 1xPBS.

·         Permeabilise in PBT (PBS+0.3%triton) for 30 minutes.

·         Block with PAT [=PBT+10% goat serum ] at RT for 3 hours.

·         Incubate o/n at 4°C with 1° Antibody diluted in PAT.

Or

             Heat fixation

·         Put E-Wash (10mM NaCl, 0.1% Triton) at 80°C on heating block for about 20 minutes. Make 100µl aliquots (1aliquot=1 dish) in small eppendorfs to put on heating block.

·         Fix for 8 seconds in E-wash at 80°C. Remove PBS from dishes and add 100µl E-wash from 80°C and put them on heating block for exactly 8 sec. Do one dish at a time.

·         Have some chilled E-wash ready in a falcon tube and pour this on the dish after 8 sec. on the heating block. Incubate the tubules on ice for about 2 minutes.

·         Remove all E-Wash and pour some methanol to cover the bottom surface of the dish. 2 hrs.

·         Remove methanol and rinse once with PBT. Wash with PBT 2x30 mins. on shaker.

·         Incubate o/n at 4°C with 1° Antibody diluted in PBT+4% goat serum.

Day 2:

·         Give 3x15minute washes with PBT (4x1hr with PBT for heat fixation protocol).

·         Blocked with PAT, 2-4 hours (PBT+4% goat serum for heat fixation).

·         Incubate o/n with 2° Antibody diluted in PAT (PBT + 4% goat serum for heat fixation) at 4°C. Cover it in an aluminium foil.

Day 3:

·         Give washes with PBT every 15 minutes for 2 hours (3x1hr washes for heat fixation protocol).

·         Incubate with DAPi for 1 min.

·         Wash with PBS (3x15 mins.).

·         Mount in vectashield.

·         Parafilm the plates and put in fridge.