May 11, 2026
Unbiased Stereology of SatB2+ neurons in M2 mouse cortex (MBF Bioscience, StereoInvestigator Software)
- Jhodi Webster1,
- Ashley Harms1,
- Asta Zane1,
- Marissa Menard1
- 1University of Alabama at Birmingham
- Marissa Menard: Editor of forked version
Protocol Citation: Jhodi Webster, Ashley Harms, Asta Zane, Marissa Menard 2026. Unbiased Stereology of SatB2+ neurons in M2 mouse cortex (MBF Bioscience, StereoInvestigator Software). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4zqrlx1/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: May 07, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316602
Keywords: ASAPCRN, unbiased stereo logical assessment of ne..., stereoinvestigator software, using stereoinvestigator software, unbiased stereology, mbf bioscience, specific brain region, other cells in specific brain region, neuron, mouse cortex, cell counting, secondary motor cortex, unbiased stereology of satb2, m2 mouse cortex, neurons in m2 mouse cortex, m2 cortex, secondary motor cortex, stereology protocol, neurons as the marker, mouse brain tissue, unbiased stereology, vi of the secondary motor cortex, satb2, stereoinvestigator software, neuron, mbf bioscience, objects in an unbiased fashion, sampling region
Funders Acknowledgements:
ASAP CRN
Grant ID: ASAP2024
Abstract
This adapted stereology protocol allows users to count objects in an unbiased fashion in layers V/VI of the secondary motor cortex (M2 cortex) of mouse brain tissue. This particular protocol uses SatB2+ neurons as the marker of interest but could be used to count a variety of markers in the M2 cortex. Changes were made to the origin protocol to account for differences in sampling region and marker of interest, other minor changes were made for clarity.
Guidelines
| A | B | C | D | |
| BRAIN REGION (mouse) | COUNTING FRAME | GRID SIZE | SECTION # (to count) | |
| M2 Cortex | 40μm x 40μm | 200 x 200 | 6 | |
| Striatum | 40μm x 40μm | 300 x 300 | 6 | |
| Hippocampus | 30μm x 30μm | 130 x 130 | 5-7 | |
| SNpc | 50μm x 50μm | 100 x 100 | 5-7 |
The table above describes the set parameters for counting cells within different brain regions. Note that evaluation interval (set in the first step of the workflow) should be based on how tissue was sectioned. For whole brain collected in 12-well plate: interval = 12, for brain sections collected in 6-well plate: interval = 6. Confidence interval should be less than 0.1.
Example of contour drawn to delineate ipsilateral M2 cortex in coronal mouse brain section.
Example of contour drawn to delineate contralateral M2 cortex in coronal mouse brain section.
Before start
This protocol was written for use with 40 µm coronal mouse brain sections. Sections of M2 cortex were selected between AP+2.5mm and AP+1.0mm. Sections were stained with SatB2 antibody #ab92446 from abcam at 1:1000 and then detected with a biotinylated secondary, HRP conjugated ABC kit and DAB chromogen deposition.
WORKFLOW
Create a map of your slide using the stereology worksheet and indicate which sections will be counted. Ensure you can differentiate between ipsilateral and contralateral hemispheres if necessary.
Set slide properly on the microscope. Set a reference point anywhere on the slide.
Click on Probes > Optical Fractionator Workflow > Start a new subject
- Label subject/study
- Existing sample in configuration: no
- Number of sections to count: 6
- Enter cut thickness: 40μm
- Section Evaluation Interval: 6* (See Guidelines & Warnings)
- Start with number one
- z-value of first section = 0
Select Low Mag Lens, click 4X > Next Step
Trace the Contours of Interest for each section on your slide. If you will be differentiating between hemispheres, choose separate contours to indicate left from right and rename the contour styles to reflect which is which. → See Guidelines and Warning for example images of boundaries for layer V/VI or M2 cortex.
Select High Mag Lens select 40X (use light focuser/lens condenser if using an upright scope) > Next Step
Select 'manually enter the average mounted thickness'. Average Mounted thickness is 33 μm
Define the counting frame: 40 µm x 40 µm → Force Counting Frame to be square
Goal is to be able to see ~5-10 of the cells you are counting per frame.
Manually enter grid size (SRS Grid Layout): 200 x 200
This defines how close your counting frames are to each other (smaller = visit more sites)
Optical Director Height > 22 µm
Count objects: Select your first section > Select the desired region > Start counting
If you have different contours for "left" and "right" also ensure that you select a distinct marker for each side.
Focus on the top of the tissue > OK
Do this by focusing at any z-plane and then adjusting the fine focus so that you are moving "up" until you are slightly out of focus - this is the "top".
Select your markers according to the desired cell types (these can also be customized)
- For each site, scroll up and down throughout the z plane and click on cells to add a marker
- Count cells with visible nucleus in the square. If a cell touches a line of the counting frame: Touching the green line: count the cell; if it touches a red line, do NOT count it.
Once you have selected all of the contours and visited all sites: Select "Completed Counting" to view results and export data to excel: View Results > Export to Excel
Ensure coefficients of error fall below the accepted threshold, for us that was 0.12
To begin a new slide/animal, go to the "New" icon in the top left corner and repeat steps.