TITLE: Immunofluorescent Labelling of Post-Mortem Rodent Brain Tissue
AUTHORS: Jeffrey  Stedehouder, Shinil Raina, Stephanie J Cragg

[DESCRIPTION]
This protocol is to label one or several targets using a two-step immunofluorescent approach in PFA perfusion-fixed, 50-µm thick, post-mortem rodent brain tissue. 
 


[GUIDELINES]
Every antibody and target has its own optimal binding conditions. Several portions of this protocol could be optimized for specific labelling targets.

[STEPS]
SECTION: Washes
1. Place 50 µm thick brain slices in a jar (or well). The jar or well should be able to hold ~2-5 ml Phosphate Buffered Saline (PBS) for proper washing.
 



SECTION: Washes
3. Place the jar or well on an orbital shaker for ~10-15 mins. 
 

SECTION: Preincubation
6. Prepare preincubation solution containing PBS with 10% Normal Bovine Serum (NBS) and 0.5% Triton-X (total 1.5 ml per well/jar) during the last wash.
SECTION: Preincubation
7. Remove the PBS from the last wash and add the preincubation solution. 
 

SECTION: Primary Antibody
10. Add the Primary Antibody to the above mix in the desired concentration. For instance, the S100β and ChAT antibodies were used at 1:1000 and 1:100, respectively. 
SECTION: Washes
13. Remove all the Primary Antibody Solution and add fresh PBS.
SECTION: Secondary Antibody
17. Prepare PBS with 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the last wash. 
SECTION: Secondary Antibody
18. Add the Secondary Antibody to the above mix in the desired concentration (commonly 1:300). For instance, Donkey anti-Guinea pig Alexa Fluor 488 and Donkey anti-Goat Dylight 594 were used at 1:300. 
SECTION: Secondary Antibody
19. Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution. 
 

SECTION: Secondary Antibody
20. Leave to incubate for ~2-5 h at room temperature on an orbital shaker.
SECTION: DAPI
25. Prepare DAPI solution by mixing 1 µl DAPI Readymade solution in 1 ml PB (1:1000). 
SECTION: DAPI
26. Remove the PB from the last wash and add the DAPI Solution.
SECTION: Washes
28. Remove all the DAPI Solution and add fresh PB. 
SECTION: Mounting
34. Add nail polish to the edges to seal the Vectashield shut and leave to dry before long-term storage in the fridge.
 

SECTION: Washes
4. After ~10-15 mins, remove the majority of PBS and add fresh PBS. 
SECTION: Washes
5. Repeat wash (steps 3 and 4) two more times for 10-15 mins each. 
SECTION: Preincubation
8. Leave to preincubate at room temperature for 1 h on an orbital shaker.
SECTION: Primary Antibody
12. Leave to incubate for ~48-72 h at 4º C on an orbital shaker.
SECTION: Primary Antibody
9. Prepare 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the preincubation period. 
SECTION: Washes
2. Add a volume (~2 ml) of PBS in each jar (or well). 
 

SECTION: Washes
14. Place the jar or well on an orbital shaker for ~10-15 mins. 
 

SECTION: Washes
15. After ~10-15 mins. remove the majority of PBS and add fresh PBS. 
SECTION: Washes
16. Repeat wash (steps 14 and 15) two more times for 10-15 mins each. 
SECTION: DAPI
27. Leave on the orbital shaker for ~15 mins.
 

SECTION: Washes
24. Repeat wash (steps 22 and 23) one more time for ~10 mins. 
SECTION: Washes
23. After ~10 minutes, remove the majority of PB and add fresh PB. 
SECTION: Washes
22. Place the jar or well on an orbital shaker for ~10 mins. 
 

SECTION: Washes
21. Remove all the Secondary Antibody Solution and add fresh Phosphate buffer (PB). 
SECTION: Washes
29. Place the jar or well on an orbital shaker for ~10 mins. 
 

SECTION: Washes
30. After ~10 minutes, remove the majority of PB and add fresh PB. 
SECTION: Washes
31. Repeat wash (steps 29 and 30) one more time for ~10 mins. 
SECTION: Mounting
33. Apply a small drop of Vectashield on the glass coverslip, and carefully place the coverslip on the glass slides containing the brain slices.
SECTION: Mounting
32. Mount the individual slices on a glass microscope slide.
SECTION: Primary Antibody
11. Once done, remove all the preincubation solution and add the Primary Antibody Solution.