Abstract
Background
Immunohistochemistry techniques represent a powerful tool to detect and quantify disease related proteins. Improvements were accomplished by tagged antibodies using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS). However, these approaches are effected by day-to-variations due to instrumental drift.
New Method
Brain tissue from line 62, a Parkinson’s disease model, and control mice were incubated with four antibodies relevant to the disease and standardized to three house-keeping proteins. In addition, a new standardization approach was developed and the results compared. This new approach consisted of coating specimens with gelatin and printing an indium-doped ink with a commercial ink jet printer. Furthermore, the method was evaluated for different ablation spot sizes with respect to resolution and signal-to-noise ratio.
Results
Normalization using house-keeping proteins led to high background signals even at high resolution. Normalization using indium-doped ink improved the signal-to-noise ratio even when small laser spot sizes were used and further improved by overlaying tissue specimen with gelatin.
Comparison with Existing Methods
Line 62 mice had more α-Synuclein and gliosis but decreased numbers of neurons, as found by conventional immunohistochemistry. These data are in line with the results obtained by LA-ICP-MS with indium standardization. However, differences between L62 and controls for tyrosine hydroxylase were only detected by LA-ICP-MS.
Conclusions
Internal standardisation using indium-doped inks is an effective method to overcome day-to-day variations and instrumental drifts. The new approach results in an increased signal-to-noise ratio and only under these conditions small but significant changes were detected, as seen for tyrosine hydroxylase.
Immunohistochemistry techniques represent a powerful tool to detect and quantify disease related proteins. Improvements were accomplished by tagged antibodies using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS). However, these approaches are effected by day-to-variations due to instrumental drift.
New Method
Brain tissue from line 62, a Parkinson’s disease model, and control mice were incubated with four antibodies relevant to the disease and standardized to three house-keeping proteins. In addition, a new standardization approach was developed and the results compared. This new approach consisted of coating specimens with gelatin and printing an indium-doped ink with a commercial ink jet printer. Furthermore, the method was evaluated for different ablation spot sizes with respect to resolution and signal-to-noise ratio.
Results
Normalization using house-keeping proteins led to high background signals even at high resolution. Normalization using indium-doped ink improved the signal-to-noise ratio even when small laser spot sizes were used and further improved by overlaying tissue specimen with gelatin.
Comparison with Existing Methods
Line 62 mice had more α-Synuclein and gliosis but decreased numbers of neurons, as found by conventional immunohistochemistry. These data are in line with the results obtained by LA-ICP-MS with indium standardization. However, differences between L62 and controls for tyrosine hydroxylase were only detected by LA-ICP-MS.
Conclusions
Internal standardisation using indium-doped inks is an effective method to overcome day-to-day variations and instrumental drifts. The new approach results in an increased signal-to-noise ratio and only under these conditions small but significant changes were detected, as seen for tyrosine hydroxylase.
| Original language | English |
|---|---|
| Article number | 108591 |
| Number of pages | 10 |
| Journal | Journal of Neuroscience Methods |
| Volume | 334 |
| Early online date | 8 Jan 2020 |
| DOIs | |
| Publication status | Published - 15 Mar 2020 |