TITLE: Immunofluorescence for confocal imaging after slice recording
AUTHORS: Cecilia Tubert, DeNard V Simmons

[DESCRIPTION]
This protocol describes the steps for immunostaining and confocal imaging of ex vivo slices following an electrophysiological experiment. 


[STEPS]
SECTION: Immunofluorescence - Before the procedure:
1. Prepare PBS

SECTION: Immunofluorescence - Before the procedure:
5. If needed, prepare aliquots of NGS. Store at -20C and thaw immediately before use.

SECTION: Immunofluorescence - Before the procedure:
6. If needed, reconstitute/aliquot antibodies and store them according to manufacturer’s instructions. Thaw them on ice immediately before use.

SECTION: Immunofluorescence - Before the procedure:
7. It is recommended to prepare the other solutions as needed

SECTION: Procedure:
8. Select the slices of interest and transfer them in a new plate with PBS.

SECTION: Procedure:
9. For better results when performing an immunostaining targeting an epitope contained in subcellular organelles, we had better results performing a short permeabilization stage with a higher concentration of detergent. This step is however optional.

SECTION: Procedure:
10. Prepare permeabilization solution: 0.5% Triton in PBS. The desired volume of permeabilization solution depends on the number of slices to process, the size of the wells/volume of solution needed to cover the slices. For a 24 wells plate, we recommend 1 ml/well of solution for permeabilization/PBS wash/blocking steps, but this can be decreased to ~300ul/well for incubation with antibodies.

SECTION: Procedure:
11. Remove PBS from each well containing a slice and substitute with permeabilization solution.

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12. Incubate for 15 mins with constant gentle movement over orbital shaker.

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13. While the slices are incubating with permeabilizing solution, prepare blocking solution (e.g.: 10% NGS, 0.25% Triton-X100 in PBS). This is the solution where the primary antibody/antibodies will be diluted, so when calculating the required volume account for the volume required for both steps (e.g.: 1ml + ~300ul per well).

SECTION: Procedure:
14. After the 15 minutes, remove permeabilization solution, rinse with PBS, and cover slices with blocking solution.

SECTION: Procedure:
15. Incubate for 30 mins with constant gentle movement over orbital shaker.

SECTION: Procedure:
16. During the incubation, dilute the primary antibodies in blocking solution according to recommended dilution.

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17. After the 30 mins incubation, remove blocking solution and apply diluted primary antibody/antibodies.

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18. Incubate over gentle agitation. Depending on the quality of the antibody used, it can be recommended to incubate the slices with the primary antibody solution over-night at 4°C, with gentle agitation.

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19. After primary antibody incubation, remove primary solution and cover slices with PBS.

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20. Wash for 10 mins over gentle agitation.

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21. Repeat wash step for a total of 3 times.

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22. During the washes, prepare the blocking solution for the secondary antibody/antibodies (e.g.: 10% NGS in PBS). Calculate needed volume based on the number of slices/size of wells and dilute secondary antibody/antibodies according to manufacturer’s instructions.

SECTION: Procedure:
23. After the PBS washes are completed, remove PBS and cover slices with the secondary antibody solution.

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24. Incubate at room temperature with constant gentle agitation on orbital shaker for 60 mins.

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25. After incubation, discard secondary antibody solution and cover with PBS.

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26. Repeat 3 10 mins PBS washes as above.

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27. After PBS washes, with a fine brush carefully mount slices on clean and labelled microscopy slides.

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28. Gently remove excess solution.

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29. Let dry in the dark (recommended: overnight).

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30. Following day/when the slices have dried on the slide: apply a small amount of hard-drying mounting medium sufficient to cover the slices. Carefully avoid the formation of air bubbles. Gently apply a coverslip over the slices and the mounting medium

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31. Let cure overnight in the dark.

SECTION: After the procedure:
32. Dispose of waste and excess reagents/solution according to institutional guidelines.

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33. Clean tools/working station.

SECTION: After the procedure:
34. Once the mounting medium is cured, slides are ready for observation.

SECTION: After the procedure:
35. Fully mounted microscopy slides should be stored in a designated container at 4C until time of observation.

SECTION: Confocal imaging - Procedure:
36. Turn on laser/microscope/computer according to specific manuals/instructions

SECTION: Confocal imaging - Procedure:
37. Access imaging software

SECTION: Confocal imaging - Procedure:
38. Load microscopy slides in the designated stage

SECTION: Confocal imaging - Procedure:
39. Proceed with observation/image acquisition according to the specific microscope/software manuals

SECTION: After the procedure:
40. Clean immersion objectives with a lens wipe and the appropriate cleaning solution

SECTION: After the procedure:
41. Export/save images

SECTION: After the procedure:
42. Turn off software/microscope/laser according to specific instructions

SECTION: After the procedure:
43. Images can be observed and appropriately adjusted using an imaging software (e.g. FIJI).

SECTION: After the procedure:
44. Copies of the original unprocessed images acquired should be stored.

SECTION: Immunofluorescence - Before the procedure:
2. Remove slice from the recording chamber.  Gently transfer into a plate and bathe with a fixative solution for further processing.
SECTION: Immunofluorescence - Before the procedure:
4. For longer storage, a preservative (e.q. sodium azide) can be added to the PBS. 
SECTION: Immunofluorescence - Before the procedure:
3. Replace fixative with PBS. Slices can be stored in the plate with PBS at 4C. It is recommended to wrap cell plate and its lid with parafilm to avoid PBS evaporation.