TITLE: Immunohistochemistry for Carbon Fiber Thread Electrodes
AUTHORS: Helen N Schwerdt, Tomoko Yoshida, Ann M Graybiel

[DESCRIPTION]
Methods for immunofluorescent staining of brain tissue with indwelling electrodes are described.

[STEPS]
1. 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.
2. Sections were left shaking for one night at 4°C in primary antibodies diluted in TSA block.
3. Primary antibody and concentration used was: rabbit anti-MOR1(Abcam Ab134054, 1:500).
4. 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.
5. 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).
6. 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.
7. 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).
8. Slides were covered with aluminum foil and stored in 4°C until imaging.
9. 100-μm sections on the glass slides from rat A were cover slipped without any staining with ProLong Gold antifade reagent.
10. 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.
11. After three times washing with PBS-Tx, slides were blocked in TSA block in the humidity-controlled chamber.
12. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. Slides were covered with aluminum foil and stored at 4°C until imaging was done.
18. The TissueFAXS Whole Slide Scanning System (TissueGnostics) with x20 objective lens was used to obtain tiled-images from rat A.
19. 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.
20. 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.