Mar 11, 2025
Histology Fushiki et al 2024
- Akira Fushiki1,2,3
- 1Allen Institute;
- 2Zuckerman Mind Brain Behavior Institute, Columbia University;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Akira Fushiki 2025. Histology Fushiki et al 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9o49v4o/v1
Manuscript citation:
A Vulnerable Subtype of Dopaminergic Neurons Drives Early Motor Deficits in Parkinson’s Disease
Akira Fushiki, David Ng, Zachary R. Lewis, Archana Yadav, Tatiana Saraiva, Luke A. Hammond, Christoph Wirblich, Bosiljka Tasic, Vilas Menon, Joaquim Alves da Silva, Rui M. Costa
bioRxiv 2024.12.20.629776; doi: https://doi.org/10.1101/2024.12.20.629776
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: December 19, 2024
Last Modified: March 11, 2025
Protocol Integer ID: 116216
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020551
Abstract
Histology protocol for Fushiki et al 2024 , to quantify Th+ cells and visualize the dorsal-tier or ventral-tier of SNc DANs.
Materials
24-well tissue culture plates
Normal Goat Serum (Abcam #ab7481) or Donkey Serum (Millipore Sigma, #S30)
PBS (prepared from tablets; Sigma, #P4417)
4% Paraformaldehyde in PBS (prepared from granules; use fresh; EMS, #19208)
1% Triton X-100 (Fisher Scientific, #BP151)
Bovine Serum Albumin (BSA; Sigma, #A2153)
Primary antibodies:
To quantify Th+ cells:
anti-GFP antibody (Chicken, Aves labs Inc, GFP-1020) or TH antibody (Mouse, ImmunoStar, 22941) at 1:2000 dilution
To visualize the dorsal-tier or ventral-tier of SNc DANs:
Calbindin D28K 1:2000 dilution (Rabbit, Synaptic Systems, 214002) and/or an Aldh1a1 antibody 1:1000 dilution (Goat, R&D Systems, AF5869)
Secondary antibodies all diluted at 1:2000 for use:
Donkey anti-Chicken Alexa Fluor 488 (Jackson ImmunoResearch, 703-545-155)
Donkey anti-Goat Alexa Fluor 568 (Invitrogen, A11057)
Donkey anti-Mouse Alexa Fluor 647 (Invitrogen, A31571)
Donkey anti-Rabbit Alexa Flour 647 (Invitrogen, A31573)
DAPI (Sigma, #D9542; 1:1000)
Used as a counterstain in all experiments
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
This protocol was approved by Columbia University IACUC. Please do not perform any of these procedures unless there is prior approval from the institution's animal ethics committee.
Before start
Read through protocol before starting.
Perfusion
Perfusion
Deeply anesthetize the mouse using 5% isoflurane.
Shave the abdominal hair and make an incision in the abdominal skin. Expose the heart by removing the rib cage, snip the right atrium, and insert a fine-gauge needle into the left ventricle.
Perform transcardial perfusion with ice-cold PBS until the fluids run clear—indicated by the liver and ears turning pale—ensuring proper perfusion.
Then, switch to fresh, ice-cold 4% paraformaldehyde and perfuse 25 mL at a flow rate of approximately 5 mL/min. During fixation, the limbs, tail, and head may move, and the mouse should become stiff if the fixation is successful.
Dissect the brain and incubate it overnight in 4% PFA at 4°C.
Prepare Tissue Sections
Prepare Tissue Sections
(Optional) Before sectioning, it is recommended to embed the brain in 4% agarose, as this helps stabilize the sample and ensures even slices. This is particularly useful when sectioning the whole brain, as certain regions, such as the caudal part of the hippocampus, may become separated during the process. The agarose helps maintain their position.
Using a Leica VT1000 vibratome, cut coronal sections at either 30 μm or 75 μm thickness, with 75 μm primarily for whole-brain 3D reconstruction. Arrange the sections sequentially in a 24-well plate with PBS for further processing.
Permeabilization
Permeabilization
Permeabilize tissue sections in PBS containing 0.4% Triton X-100 (PBST) for two rounds of 10–15 minutes each.
Blocking
Blocking
Replace PBST with blocking buffer (1x PBS, 0.4% Triton X-100, 3% BSA, and 2% Goat or Donkey Serum) and incubate at room temperature for 1 hour on a shaker.
Primary Antibody Labeling
Primary Antibody Labeling
Dilute the primary antibodies desired in incubation buffer (1x PBS, 0.4% Triton X-100, and 2% Goat or Donkey serum). Refer to the Materials section for dilution details.
Add the diluted primary antibodies to each well and incubate overnight at 4°C on a shaker, ensuring the tissue sections remain suspended in the antibody solution.
(Example) For DAT-Cre slices, use anti-TH (Mouse, ImmunoStar, #22941) at a 1:2000 dilution.
After overnight incubation, transfer the plate to the bench, replace the solution in each well with PBS, and wash twice for 30 minutes each.
Secondary Antibody Labeling
Secondary Antibody Labeling
Dilute the secondary antibody in incubation buffer (1x PBS, 0.4% Triton X-100, and 2% goat or donkey serum) as per the manufacturer's guidelines.
Add the diluted secondary antibodies to each well and incubate overnight at 4°C on a shaker, ensuring the tissue sections remain suspended in the antibody solution.
Secondary Antibody Labeling
Secondary Antibody Labeling
(Example) For DAT-Cre slices, use Donkey anti-Mouse Alexa Fluor 647 (Invitrogen, #A31571) at a 1:2000 dilution.
After overnight incubation, transfer the plate to the bench, replace the solution in each well with PBS, and wash twice for 30 minutes each.
DAPI Staining
DAPI Staining
Prepare a working DAPI solution (following the manufacturer's guidelines, Sigma, #D9542) by diluting the stock to 1:1000 in PBS.
Replace the secondary antibody solution with the DAPI solution in each well and incubate for 15 minutes at room temperature on a shaker, ensuring the plate is protected from light (typically by covering with aluminum foil).
DAPI Staining
DAPI Staining
Rinse sections with PBS for 15 minutes at room temperature on a shaker, ensuring the plate is protected from light (typically by covering with aluminum foil).
Mounting
Mounting
Transfer sections to histology slides and mount in mounting media, avoid creating bubbles and protect from light once completed.
Allow mounting media to dry before imaging.