Immunofluorescent Labelling of Post-Mortem Rodent Brain Tissue
Shinil Raina, Stephanie J Cragg, Jeff Stedehouder
Disclaimer
Special thanks to Elize Haasdijk and Erika Sabel-Goedknegt for help with very early versions of this protocol
Abstract
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.
Steps
Washes
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.
Add a volume (~2 ml) of PBS in each jar (or well).
Place the jar or well on an orbital shaker for ~10-15 mins.
After ~10-15 mins, remove the majority of PBS and add fresh PBS.
Repeat wash (steps 3 and 4) two more times for 10-15 mins each.
Preincubation
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.
Remove the PBS from the last wash and add the preincubation solution.
Leave to preincubate at room temperature for 1 h on an orbital shaker.
Primary Antibody
Prepare 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the preincubation period.
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.
Once done, remove all the preincubation solution and add the Primary Antibody Solution.
Leave to incubate for ~48-72 h at 4º C on an orbital shaker.
Remove all the Primary Antibody Solution and add fresh PBS.
Place the jar or well on an orbital shaker for ~10-15 mins.
After ~10-15 mins. remove the majority of PBS and add fresh PBS.
Repeat wash (steps 14 and 15) two more times for 10-15 mins each.
Secondary Antibody
Prepare PBS with 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the last wash.
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.
Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution.
Leave to incubate for ~2-5 h at room temperature on an orbital shaker.
Remove all the Secondary Antibody Solution and add fresh Phosphate buffer (PB).
Place the jar or well on an orbital shaker for ~10 mins.
After ~10 minutes, remove the majority of PB and add fresh PB.
Repeat wash (steps 22 and 23) one more time for ~10 mins.
DAPI
Prepare DAPI solution by mixing 1 µl DAPI Readymade solution in 1 ml PB (1:1000).
Remove the PB from the last wash and add the DAPI Solution.
Leave on the orbital shaker for ~15 mins.
Remove all the DAPI Solution and add fresh PB.
Place the jar or well on an orbital shaker for ~10 mins.
After ~10 minutes, remove the majority of PB and add fresh PB.
Repeat wash (steps 29 and 30) one more time for ~10 mins.
Mounting
Mount the individual slices on a glass microscope slide.
Apply a small drop of Vectashield on the glass coverslip, and carefully place the coverslip on the glass slides containing the brain slices.
Add nail polish to the edges to seal the Vectashield shut and leave to dry before long-term storage in the fridge.
