1. Perfuse-fix tissue in 4% paraformaldehyde in PBS (can use PLP fixative instead).
2. Vibratome 50µm sections through areas of interest.
3. Wash sections 3 x 5 min in PBS.
4. Incubate sections in 1% sodium borohydridea in PBS for 10 min.
5. Wash sections 3 x 5 min in PBS.
6. Block sections in 10% normal serumb + 0.5% BSA + 0.5% Triton X-100 in PBS for 30 min.
7. Rinse sections in PBS.
8. Incubate sections in primary antibodies, diluted appropriately in 1% normal serum + 0.5% BSA + 0.5% Triton X-100 in PBS, overnight.
9. Wash sections 3 x 5 min in PBS.
10. Incubate sections in secondary antibodies (AlexaFluor 488, 546 and 633 conjugatesc), diluted 1:750 in 1% normal serum + 0.5% BSA + 0.5% Triton X-100 in PBS, for a minimum of 2 hr in dark.
11. Wash sections 3 x 5 min in PBS in dark.
12. Either, mount in VectorShield, coverslip and seal with nail varnish, or air-dry sections on clean slides overnight in dark, clear briefly in xylene (30 sec) and mount in Fluoromountd.
13. View in confocal using multi-tracking protocole.

Triple-Labelling Immunofluorescence Protocol (Cell Cultures)

1. Culture cells on coverslips.
2. Rinse coverslips briefly in medium (without serum).
3. Fix cells in 4% paraformaldehyde in PBS for 30 min.
4. Wash cells 3 x 5 min in PBS.
5. (Optional) Incubate sections in 0.3M glycine in PBS for 10 min.
6. Wash cells 3 x 5 min in PBS.
7. If required, permeablise cells by incubating in 0.5% Triton X-100 in PBS for 10 min.
8. Wash cells 3 x 5 min in PBS.
9. Block cells in 10% normal serumb + 0.5% BSA in PBS for 30 min.
10. Incubate sections in primary antibodies, diluted appropriately in 1% normal serum + 0.5% BSA in PBS, for a minimum of 2 hr.
11. Wash cells 3 x 5 min in PBS.
12. Incubate cells in secondary antibodies (AlexaFluor 488, 546 and 633 conjugatesc), diluted 1:750 in 1% normal serum + 0.5% BSA in PBS, for a minimum of 1 hr in dark.
13. Wash cells 3 x 5 min in PBS in dark.
14. Mount in VectorShield on a clean slide and seal with nail varnish.
15. View in confocal using multi-tracking protocole.
Notes
a. Sodium borohydride lowers background fluorescence by reducing free aldehyde groups. It can be damaging to tissues due to the release of hydrogen gas, so monitor the reaction. Also, because of this effect, it is not routinely used on cell cultures.
b. Normal serum should be from secondary antibody host species. If possible, use the same host species for all secondary antibodies (typically goat). If this is not possible, choose the host species carefully to avoid cross-reactivity with other antibodies.
c. Other AlexaFluor dyes may be used (for instance AlexaFluor 594 instead of 546). If using a different dye, it may be necessary to alter the appropriate filter set on the multitracking settings. If this is done, please save the settings under a new name!
d. VectorShield mounting consistently gives the best results. However, for a permanent preparation, Fluoromount is preferred. The xylene can be detrimental to the fluorochromes so only a brief clearing step is advised.
e. A multi-tracking protocol for detecting AlexaFluor 488, 546 and 633 triple-labelled samples has been programmed into the confocal. Simply select multi-Track?in the configuration?window and then choose the triple labelling settings from the drop-down menu. Essentially, the rest is the same. One thing to check is that the pin-hole size for each track is different (it is best to set each pinhole to 1 airy unit).