Jan 10, 2024
Histology, Immunohistochemistry and Imaging
- Jonathan Tang1,2,3
- 1Department of Neuroscience, Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10027, USA;
- 2Seattle Children’s Research Institute, Center for Integrative Brain Research, Seattle, WA, USA;
- 3University of Washington School of Medicine, Department of Pediatrics, Seattle, WA, USA
Protocol Citation: Jonathan Tang 2024. Histology, Immunohistochemistry and Imaging . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjbdelx9/v1
Manuscript citation:
Tang, J.C.Y., Paixao, V., Carvalho, F. et al. Dynamic behaviour restructuring mediates dopamine-dependent credit assignment. Nature (2023). https://doi.org/10.1038/s41586-023-06941-5
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: 93347
Keywords: ASAPCRN
Funders Acknowledgement:
Life Sciences Research Fellowship
NINDS K99/R00 Award
Grant ID: 1K99NS112575
NIH
Grant ID: 5U19NS104649
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020551
Abstract
Sample preparation, histology, immunohistochemistry (IHC) and imaging of brain sections in Tang et al 2023.
Materials
Standard perfusion setup
Vibrotome
Zeiss Axio ImagerM2 microscope and accessories (see steps for exact details)
Antibodies/Stains
-Rabbit anti-GFP 488 conjugate (1:1000;Molecular Probes A21311, RRID:AB_221477)
-Mouse Anti-TH (1:5000;Immunostar Th 22941, RRID:AB_572268)
-Goat Anti-Mouse - IgG (H+L) Highly cross-adsorbed secondary antibody - Alexa Fluor647(1:1000; ThermoFisher, A-21236, RRID:AB_2535805)
-DAPI (1:1000 of 20 mg/mL stock; Sigma, D9542).
Before start
Process begins after behavioral sessions were completed.
Sample Preparation
Sample Preparation
Deeply anesthetized mouse with isoflurane.
Perfuse transcardially in PBS and then 4% PFA/PBS.
Dissect brains with skulls attached and submerged in 4% PFA in PBS at 4 degrees Celsius overnight
Next day-brains were rinsed 3 times in PBS.
Brain regions including VTA and implants were sectioned by vibratome into 50 or 100 μm slices (depending on desired output).
Histology/Immunohistochemistry
Histology/Immunohistochemistry
Slices were stained with the following reagents by the Champalimaud Histopatholgy Platform (core facility) using their standard protocol.
-Rabbit anti-GFP 488 conjugate (1:1000;Molecular Probes A21311, RRID:AB_221477)
-Mouse Anti-TH (1:5000;Immunostar Th 22941, RRID:AB_572268)
-Goat Anti-Mouse - IgG (H+L) Highly cross-adsorbed secondary antibody - Alexa Fluor647(1:1000; ThermoFisher, A-21236, RRID:AB_2535805)
-DAPI (1:1000 of 20 mg/mL stock; Sigma, D9542).
Imaging
Imaging
Using a Zeiss Axio Imager M2 microscope-10x tiled images were taken through the relevant fluorescent channels.
The M2 is equipped with a fast Colibri.7 LED illumination for excitation of fluorophores. Images are captured with a high-sensitivity monochromatic sCMOS camera(Hamamatsu Orca Flash 4.0 v2). The objective used for the images is a ZEISS Plan-ApoChromat 10x/0.45, which allowsto resolve up to 577 nm when using a wavelength of observation of 520nm and it is fully corrected for chromatic and spherical aberrations. Implant locations were determined using Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates.