Mar 16, 2025
Semi-Quantitative Histology Scoring
- anahid.ansari1,
- Barkha Goswami1,
- Tony Hsiao2,
- YuHong Fu1,
- Berenice Coutant3,
- Alexandra Nelson3,
- Glenda Halliday3,
- Kasandra Romo3
- 1USTD;
- 2Usyd;
- 3UCSF
Protocol Citation: anahid.ansari, Barkha Goswami, Tony Hsiao, YuHong Fu, Berenice Coutant, Alexandra Nelson, Glenda Halliday, Kasandra Romo 2025. Semi-Quantitative Histology Scoring. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrmknlpk/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: March 11, 2025
Last Modified: March 17, 2025
Protocol Integer ID: 124209
Keywords: ASAPCRN, histology, synuclein, substantia nigra, Parkinson's Disease, dopamine
Funders Acknowledgements:
ASAP
Abstract
This protocol describes our procedure for semi-quantitative scoring of midbrain mCherry histology in a cell type specific AAV-based alpha synuclein mouse model. In this model, the midbrain of DAT-Cre animals was injected with AAV encoding Cre-dependent mCherry (control group) or asyn-IRES-mCherry (synuclein group) to achieve expression in midbrain dopamine neurons. This type of scoring was not used to quantitate any readouts of pathology, but only to include/exclude animals for further physiological and/or behavioral assays. Animals were sacrificed at different timepoints after injection of AAVs, once physiological and/or behavioral assays were completed. Data from these animals was then included or excluded based on postmortem mCherry histology. This protocol includes steps to section, immunostain, image, and score brains on a 0-5 scale. Animals with a score of 2 or more on each side of the midbrain were included for additional analysis.
Attachments
Sectioning Brains
Sectioning Brains
After completing experiments, sacrifice animals, perform transcardial perfusion with paraformaldehyde (PFA, 4% in phosphate-buffered saline, PBS), store in PFA overnight, then transfer to sucrose (30% in PBS) for additional storage, minimum overnight.
Section brains into 30 µm coronal sections on a freezing microtome.
Place every 6th section in PBS 1X in a 24-well plate for immunohistochemistry.
Store the remaining 5 sections as replicates in cryoprotection solution in a 24-well plate.
If immunohistochemistry is not performed the same day, cover the PBS plate and store at 4°C overnight. Store the cryoprotection plate in a -20°C freezer.
To cover the entire SNc/VTA region (Bregma -3.88 mm to -2.54 mm), approximately 7 good slices are required.
Immunostaining
Immunostaining
Select your sections if necessary.
Wash sections 3 times with PBS 1X to remove any debris or residual solutions.
Incubate sections in 3% Normal Donkey Serum (NDS) with 0.1% Triton X-100 (NDST) in PBS 1X to block nonspecific binding sites.
Prepare primary antibodies in 3% NDS and PBS 1X: Rabbit anti-mCherry (1:500, M11217, Rat anti-mCherry clone 16D7); Rat anti-alpha-synuclein (1:300, ALX-804-258L001, Enzo Life Sciences); Chicken anti-tyrosine hydroxylase (TH) (1:1000, P40101-150, Pel-Freez Biologicals).
Incubate sections with the antibody mix on a shaker at 4°C for at least 1–2 days.
Wash sections 3–5 times in PBS 1X for 10 minutes each on a shaker at room temperature.
Prepare secondary antibodies in 3% NDS and PBS 1X: 568-conjugated AffiniPure Donkey Anti-Rabbit (1:300, 712-575-150, Jackson ImmunoResearch Laboratories) for mCherry; 488-conjugated AffiniPure
Donkey Anti-Rat (1:300, 712-545-150, Jackson ImmunoResearch Laboratories) for alpha-synuclein; 647-conjugated AffiniPure Donkey Anti-Chicken (1:500, 703-605-155, Jackson ImmunoResearch Laboratories) for TH.
Incubate sections with the secondary antibody mix on a shaker at 4°C for 2 days.
Wash sections 3–5 times in PBS 1X for 10 minutes each on a shaker at room temperature.
Mounting and Imaging
Mounting and Imaging
Mount sections onto slides using Vectashield hardset mounting medium with DAPI.
Allow sections to dry in the dark.
Image all sections using a Zeiss Axioscan at 20X epifluorescence with 4 channels for DAPI, Alexa 568, Alexa 488, and Alexa 647.
Maintain consistent lighting and exposure settings for reliable comparisons and subsequent analysis.
Scoring
Scoring
Two independent raters score each slide. Raters are blinded to whether the sections are from an animal treated with AAV-DIO-mCherry (control group) or AAV-DIO-asyn-mCherry (synuclein group).
Use software capable of opening images and adjusting contrast (e.g., Zeiss Microscopy Software or QuPath-0.5.1).
Open images with the software and adjust the intensity of mCherry staining for visualization.
Assess the percentage of neurons expressing mCherry in midbrain regions (VTA and SNc) out of the total presumed dopamine (TH-expressing) neurons.
Assign scores per side: 1 = 0–20%; 2 = 20–40%; 3 = 40–60%; 4 = 60–80%; 5 = 80–100%.
Repeat scoring on each selected section for the mouse.
Average the scores for each side.
Inclusion/Exclusion
Inclusion/Exclusion
A minimum score of 2 (20-40% of cells expressing mCherry) bilaterally is required for inclusion.
Mice with asymmetric scores (difference > 2 between sides) are excluded.
Compare results between two raters and discuss discrepancies.