Jan 10, 2024
Neuromelanin Processing and Image Acquisition
- Anastasia Filimontseva1,2,
- Glenda Halliday1,2,
- YuHong Fu1,2
- 1Brain and Mind Centre & Faculty of Medicine and Health School of Medical Sciences, The University of Sydney, Sydney, NSW 2050, Australia;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Anastasia Filimontseva, Glenda Halliday, YuHong Fu 2024. Neuromelanin Processing and Image Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd799lmk/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: January 10, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93362
Keywords: ASAPCRN
Abstract
Protocol for preparing post-mortem tissue for intracellular NM quantification.
Tissue Processing
Tissue Processing
Formalin-fixed paraffin-embedded tissue blocks from the pontine and midbrain regions were sectioned at 6µm, collected onto adhesive microscope slides and allowed to dry in the oven at 37°C for 48 hours.
Slides were incubated in the oven at 60°C for 1 hour to melt the paraffin.
To remove the paraffin, sections were submerged in HistoChoice Clearing Agent (Sigma-Aldrich, H2779) for 2 x 7 minutes, followed by rehydration in decreasing ethanol concentrations (100% ethanol for 2 x 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes) and distilled H2O for 3 minutes.
Slides were submerged in 1xPBS and coverslipped using Fluorescence Mounting Medium (DAKO, S302380-2).
Image Acquisition
Image Acquisition
Sections were scanned using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal NM signal quality with an automated Slide Scanner (SLIDEVIEW VS200, Tokyo, Japan).
Identical parameters were applied to each scanned section to ensure consistency in capturing NM.
Once the scan was completed, sections were submerged in 1xPBS to remove coverslips and continue with IF processing.