TITLE: Proteinase K digestion
AUTHORS: Martin T. Henrich, Fanni F. Geibl

[DESCRIPTION]
This protocol describes the steps for performing proteinase K digestion and subsequent visualization by immunofluorescence, which can be helpful in determining insolubility of alpha-synuclein aggregates.




[BEFORE_START]
-Prepare 0.1 M PB (PB)

-Prepare PBT (0.1 M PB with 0.3% Triton X-100)

-Prepare NDS solution (10% normal donkey serum diluted in PBT)

-Prepare proteinase K solution (12 µg/ml in 0.1 M PB) 

-Set water bath to 65°C

[STEPS]
SECTION: Proteinase K digestion - Procedure
1. -Place individual sections in 12-well-plate with netwell inserts in PB.
SECTION: Proteinase K digestion - Procedure
4. -Perform PK digestion by immediately putting the filled 12-well-plate in the water bath (65 °C) followed by 10 min shaking.65 °C 

SECTION: Proteinase K digestion - Procedure
2. -Wash sections for 3x 5 min on platform shaker in PB, change solution between washing steps.
SECTION: Proteinase K digestion - Procedure
3. -Place sections in 12-well-plate with netwell inserts in PK solution. Use 4 ml solution per well.  
SECTION: Proteinase K digestion - Procedure
5. -Take well-plate out of the water bath and wash sections 4x 5 min on platform shaker
in 0.1 M PB at room temp., change solution between washing steps.

Room temperature 


SECTION: Proteinase K digestion - Procedure
6. -Block sections for 60 min at room temp. in NDS solution in 12-well-plates. We recommend 4 ml solution per well for good results. 
SECTION: Proteinase K digestion - Procedure
7. -For incubation with primary antibodies, transfer sections in a new 12-well-plate
but without netwell inserts. This allows better shaking overnight. Solution for
incubation with primary antibodies should contain NDS solution and the
respective primary antibodies diluted according to manufacturer recommendation.
Incubate sections on orbital shaker with gentle shaking at 4°C overnight (time may vary). We
recommend at least 1 ml solution for each well. 4 °C 

SECTION: Proteinase K digestion - Procedure
8. -On the next day, transfer sections into 12-well-plate with netwell inserts and wash
sections 4x 5 min in PBT on platform shaker at room temperature. Change washing
solution after each washing step.
SECTION: Proteinase K digestion - Procedure
9. -Next, transfer sections back in 12-well-plate without netwell inserts for incubation
with secondary antibodies.
SECTION: Proteinase K digestion - Procedure
10. -Incubate sections with fluorophore-conjugated, species-specific secondary antibodies in
NDS solution for 2 hours on orbital shaker at room temperature. For dilution of
the secondary antibodies we generally use 1:500. We recommend at least 1 ml
solution for each well. Cover the plate with aluminum foil during antibody
incubation to avoid light exposure.Room temperature 

SECTION: Proteinase K digestion - Procedure
11. -Thereafter, transfer sections back to 12-well-plate with netwell inserts and wash sections
for 5x 5min in PBT. For all washing steps, 12-well-plate should be
covered with aluminum foil to avoid light exposure.



SECTION: Proteinase K digestion - Procedure
12. -After washing, mount sections on microscopy slides using a fine brush.
SECTION: Proteinase K digestion - Procedure
13. -Let sections dry a few minutes.
SECTION: Proteinase K digestion - Procedure
14. -Apply a small amount of hard-drying mounting medium sufficient to cover the sections.
Carefully avoid the formation of air bubbles. Gently apply a coverslip over the sections and the mounting medium.
SECTION: Proteinase K digestion - Procedure
15. -Let cure overnight in the dark at room temperature.Room temperature 

SECTION: Proteinase K digestion - After the Procedure
16. -Dispose of waste and excess reagents/solution according to institutional guidelines.
SECTION: Proteinase K digestion - After the Procedure
17. -Clean tools/working station.
SECTION: Proteinase K digestion - After the Procedure
18. -Once the mounting medium is cured, slides are ready for observation.
SECTION: Proteinase K digestion - After the Procedure
19. -Fully mounted microscopy slides should be stored in a designated container at 4°C until time of observation.4 °C