Boris Neumann1, Simone Hösl2, Karima Schwab3, Franz Theuring4, Norbert Jakubowski5. 1. Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Center for Cardiovascular Research, Institute of Pharmacology, Hessische Strasse 3-4, 10115 Berlin, Germany; Proteome Factory AG, Magnusstrasse 11, 12489 Berlin, Germany. 2. Proteome Factory AG, Magnusstrasse 11, 12489 Berlin, Germany. 3. Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Center for Cardiovascular Research, Institute of Pharmacology, Hessische Strasse 3-4, 10115 Berlin, Germany. Electronic address: karima.schwab@charite.de. 4. Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Center for Cardiovascular Research, Institute of Pharmacology, Hessische Strasse 3-4, 10115 Berlin, Germany. 5. Spetec GmbH, Berghamer Str. 2, 85435 Erding, Germany.
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.
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.
Authors: Anna Šindelářová; Pavel Pořízka; Pavlína Modlitbová; Lucie Vrlíková; Kateřina Kiss; Milan Kaška; David Prochazka; Jakub Vrábel; Marcela Buchtová; Jozef Kaiser Journal: Sensors (Basel) Date: 2021-01-29 Impact factor: 3.576