Dec 07, 2023
Automated Immunohistochemistry Staining
- 1Department of Clinical and Movement Neurosciences, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20 815, USA;
- 3Queen Square Brain Bank for Neurological Disorders (QSBB), 1 Wakefield Street, London WC1N 1PJ, UK
Protocol Citation: Toby J Curless, Hemanth Ramesh Nelvagal, Zane Jaunmuktane 2023. Automated Immunohistochemistry Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qjz2vo5/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: August 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86041
Keywords: ASAPCRN, Immunohistochemistry, Brain sections, Ventana Discovery® XT Immunostainer, Antibody diluent solution (Ventana)
Funders Acknowledgement:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Abstract
The protocols describes the steps for automated immunohistochemistry staining using the Ventana Discovery® XT
Immunostainer.
Materials
- Primary AT8 antibody (Thermo Fisher Scientific, MN1020)
- Antibody diluent solution (Ventana)
- Ventana Discovery® XT Immunostainer: https://www.medicalexpo.com/prod/roche/product-71020-527513.html
- Leica CV5030 automated slide cover-slipping machine
- 100 % ethanol
- Oven
Preparation
Preparation
1h
Heat tissue dry tissue sections for 01:00:00 at 60 °C
1h
Generate tissue sections using standard microtome sectioning protocols.
Prepare primary and secondary antibody solutions as per manufacturer's protocol - see below for example for AT8.
Primary AT8 antibody (Thermo Fisher Scientific, MN1020) diluted at 1:100 in antibody diluent solution
(Ventana). Secondary anti-mouse antibody (Abcam) diluted at 1:100 in antibody diluent solution (Ventana).
Perform immunohistochemical staining using the Ventana Discovery® XT Immunostainer – following manufacturers guidelines.
Print barcoded slide labels corresponding to the correct protocol on the Ventana machine and stick them to the top of each slide following the slide heating.
Processing Slides Using the Ventana Machine
Processing Slides Using the Ventana Machine
25m
Place slides into the Ventana machine and ensure all bulk reagents are sufficiently filled
Begin the staining protocol.
Upon completion of the staining, activate the counterstaining step.
Following counterstaining, remove the slides and wash in soapy water 5x.
Leave slides in running water for 00:05:00 mins.
5m
To deparaffinise sections, place slides in 100 % ethanol for 00:05:00 mins, remove and place in separate 100 % ethanol for 00:05:00 mins. Remove from ethanol and place in xylene for 00:05:00 mins, remove and place in separate xylene for00:05:00 mins
20m
For cover-slipping of slides following deparaffinisation – use the Leica CV5030 automated slide cover-slipping machine.
Stained slides digitised on a NanoZoomer S360 scanner (Hamamatsu) – Brightfield scan profile at x40 magnification.
Upload digitised slides to NZ Connect (1.0.36 (IVD)) slide viewing platform.
AT8 staining
Alpha-synuclein staining