Aug 24, 2023
Immunohistochemistry for Carbon Fiber Thread Electrodes
- Helen N Schwerdt1,2,
- Tomoko Yoshida3,
- Ann M Graybiel3
- 1Department of Bioengineering, University of Pittsburgh;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD;
- 3McGovern Institute and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology
Protocol Citation: Helen N Schwerdt, Tomoko Yoshida, Ann M Graybiel 2023. Immunohistochemistry for Carbon Fiber Thread Electrodes. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpx78l5b/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: April 11, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80335
Keywords: ASAPCRN
Funders Acknowledgement:
NIH NINDS R00
Grant ID: NS107639
BBRF NARSAD Young Investigator Grant
Grant ID: 28690
Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020-519
NIH NIMH R01
Grant ID: R01 MH060379
Saks Kavanaugh Foundation
William N. and Bernice E. Bumpus Foundation
Abstract
Methods for immunofluorescent staining of brain tissue with indwelling electrodes are described.
Free floating 25-μm sections from rat A were rinsed three times for 5 min each with 0.01 M phosphate buffered saline with 0.2% Triton X-100 (PBS-Tx), and then blocked for 60 min in TSA Blocking reagent (Akoya science FP1012) diluted in PBS-Tx (TSA block) on a shaker.
Sections were left shaking for one night at 4°C in primary antibodies diluted in TSA block.
Primary antibody and concentration used was: rabbit anti-MOR1(Abcam Ab134054, 1:500).
Sections were rinsed three times for 5 min each with PBS-Tx and then were incubated in secondary antibodies diluted in TSA block for 2 hours on the shaker at room temperature.
Secondary antibodies and concentrations used were: goat anti-rat conjugated with AlexaFluor 546 (Invitrogen A-11081, 1:300), and goat anti-rabbit conjugated with AlexaFluor 647 (Invitrogen A-21245, 1:300).
Sections were rinsed three times for 5 min in 0.1 M PB, then incubated for 2 min in DAPI (Invitrogen 62248, 1:1000) diluted in PBS.
After rinsing three times for 5 min in 0.1 M PB, all sections were mounted onto glass slides and cover slipped with ProLong Gold antifade reagent (Invitrogen, P36930).
Slides were covered with aluminum foil and stored in 4°C until imaging.
100-μm sections on the glass slides from rat A were cover slipped without any staining with ProLong Gold antifade reagent.
The 100-μm sections on the glass slides from rat B were rinsed three times for 5 min each with 0.01 M PBS-Tx, and blocked endogenous peroxidase activity with 3% H2O2 in PBS-Tx.
After three times washing with PBS-Tx, slides were blocked in TSA block in the humidity-controlled chamber.
After removing the excess liquid from slides, rabbit anti-MOR1 (Abcam Ab134054, 1:500) antibody diluted in TSA block was applied on the slides and slides were left for one night at 4°C in a humidity-controlled chamber.
Slides were then rinsed three times for 5 min each with PBS-Tx and were incubated with goat anti-rabbit antibody conjugated with polymer HRP (Thermofisher B40962) for 45 minutes at room temperature.
After rinsing three times for 5 min each with PBS-Tx, slides were incubated with TSA-Plus Fluorescein (PerkinElemer NEL745001KT) diluted at 1:100 in 1X Plus Amplification Diluent for 15 minutes at room temperature.
Slides were rinsed three times for 5 min in 0.1 M PB, then incubated for 2 min in DAPI (1:1000, Invitrogen 62248) diluted in PBS.
After rinsing three times for 5 min in 0.1 M PB, slides were air dried, and then were cover slipped with ProLong Gold antifade reagent.
Slides were covered with aluminum foil and stored at 4°C until imaging was done.
The TissueFAXS Whole Slide Scanning System (TissueGnostics) with x20 objective lens was used to obtain tiled-images from rat A.
Cameras equipped with this system are Baumer HXG40c (HX series) CMOS camera 16 bit (2048 × 2048) for brightfield imaging and Hamamatsu Orca Flash 4.0 V2 cooled digital CMOS camera C11440-22CU for fluorescence imaging.
Brightfield images were obtained from 100-μm sections to see carbon fibers left in the brains and fluorescence images were obtained from 25-μm sections to visualize MOR1 enriched striosome and endogenous rat IgG.