Oct 07, 2025
Histology Albarran et al 2025
Forked from Histology Fushiki et al 2024
- Eddy Albarran1,2,3,
- Akira Fushiki1,2,3
- 1Allen Institute;
- 2Zuckerman Mind Brain Behavior Institute, Columbia University;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Eddy Albarran, Akira Fushiki 2025. Histology Albarran et al 2025. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95zeql3e/v1
Manuscript citation:
Protocol Published in:
Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons
Eddy Albarran, Akira Fushiki, Anders Nelson, David Ng, Corryn Chaimowitz, Laudan Nikoobakht, Tanya Sippy, Darcy S. Peterka, Rui M. Costa
bioRxiv 2025.06.15.659794; doi: https://doi.org/10.1101/2025.06.15.659794
Adapted from:
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: October 07, 2025
Last Modified: October 07, 2025
Protocol Integer ID: 229270
Keywords: ASAPCRN, fushiki et al 2024 histology protocol, histology protocol, histology, cell, dorsal, tier of snc dan, snc dan, albarran et al, histology albarran et al 2025 histology protocol, histology albarran et al
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020551
Abstract
Histology protocol for Albarran et al 2025 that was adapted from Fushiki et al 2024 , to quantify Th+ cells.
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
Troubleshooting
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
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
(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.
Rinse tissue in the 24-well plate with 1X PBS twice.
Permeabilization
Permeabilize tissue sections in PBS containing 0.4% Triton X-100 (PBST) for two rounds of 10–15 minutes each.
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
Dilute the primary antibodies desired in incubation buffer (1x PBS, 0.4% Triton X-100). Refer to the Materials section for dilution details.
Add the diluted primary antibodies to each well and incubate overnight at 4°C.
For Th staining, 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
Dilute the secondary antibody in incubation buffer (1x PBS, 0.4% Triton X-100).
For the anti-TH mouse primary antibody, use the appropriate secondary, for the TH antibody, use Doney anti-Mouse Alexa Fluor (Invitrogen, A31571) at a 1:2000 dilution.
Add the diluted secondary antibodies to each well and incubate overnight at 4°C.
DAPI Staining
After overnight incubation, transfer the plate to the bench, replace the solution in each well with PBS, and wash twice for 30 minutes each.
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).
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
Transfer sections to histology slides and mount in mounting media, avoid creating bubbles and protect from light once completed.
Final Steps & Imaging
Allow mounting media to dry before imaging.
Image sections using an automated slide scanner (Nikon AZ100 Multizoom microscope) equipped with a 4x 0.4NA Plan Apo objective (Nikon Instruments Inc).