Nov 22, 2023
Proteinase K digestion
- Martin T. Henrich1,2,
- Fanni F. Geibl1,2
- 1Northwestern University, Feinberg School of Medicine;
- 2Philipps University Marburg, Clinic for Psychiatry and Psychotherapy
Protocol Citation: Martin T. Henrich, Fanni F. Geibl 2023. Proteinase K digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx9qqlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 22, 2023
Last Modified: November 22, 2023
Protocol Integer ID: 91333
Keywords: ASAPCRN, Brain, Tissue digestion, Proteinase K
Funders Acknowledgement:
ASAP
Grant ID: 020551
JPB Foundation, NIH
Grant ID: NS 121174
Stichting Parkinson Fonds
Von-Behring-Röntgen Stiftung
Abstract
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.
Attachments
Materials
-Pre-cut brain sections (30-50 µm thick)
-0.1 M PB
-Triton-X100 (detergent)
-Normal donkey serum (S30-100ML, Sigma-Aldrich)
-Proteinase K (e.g. Proteinase K, Invitrogen, #4333793)
-Primary antibodies (e.g. Anti-ChAT AB144P, Merck; anti-TH ab113, abcam; anti-p62, ab109012, abcam; anti-alpha synuclein(phosphoS129), ab51253, abcam)
-Secondary antibodies
-12-well-plates with netwell inserts (e.g. Corning Costar Netwell)
-Hard-drying mounting medium (recommended ProLong Diamond, ThermoFisher Scientific)
-Aluminum foil
-Microscopy slides
-Glass coverslips
-Designated container for storage of microscopy slides
-Water bath with integrated shaker, temperature adjustable (e.g. GFL-1083)
-Orbital shaker (e.g. Heidolph Duomax 1030)
-Platform shaker (e.g. Heidolph Unimax 1010)
-Optional: DAPI, Sigma-Aldrich, D9542-5MG, 1:10,000 of 5 mg/ml
Safety warnings
Recommended PPE:
- Lab coat/disposable gown
- Safety goggles
- Examination gloves
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
Proteinase K digestion - Procedure
Proteinase K digestion - Procedure
18h 8m
-Place individual sections in 12-well-plate with netwell inserts in PB.
-Wash sections for 3x 5 min on platform shaker in PB, change solution between washing steps.
15m
-Place sections in 12-well-plate with netwell inserts in PK solution. Use 4 ml solution per well.
-Perform PK digestion by immediately putting the filled 12-well-plate in the water bath (65 °C) followed by 10 min shaking.65 °C
10m
-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
20m
-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.
1h
-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
16h
-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.
20m
-Next, transfer sections back in 12-well-plate without netwell inserts for incubation
with secondary antibodies.
-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
2h
-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.
Stain with DAPI8 steps
-In case you want to additionally stain with DAPI, perform only 2 washing steps with PBT
and add DAPI (1:10.000 of 5 mg/ml) to the third PBT washing step and let
sections wash for 10 min. Perform a final washing step without DAPI in PBT
solution. For all washing steps, 12-well-plate should be covered with aluminum
foil to avoid light exposure.
-After washing, mount sections on microscopy slides using a fine brush.
-Let sections dry a few minutes.
3m
-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.
-Let cure overnight in the dark at room temperature.Room temperature
Proteinase K digestion - After the Procedure
Proteinase K digestion - After the Procedure
-Dispose of waste and excess reagents/solution according to institutional guidelines.
-Clean tools/working station.
-Once the mounting medium is cured, slides are ready for observation.
-Fully mounted microscopy slides should be stored in a designated container at 4°C until time of observation.4 °C