Sep 13, 2022
Version 1
Stereology-mediated cell count using StereoInvestigator V.1
- Marta Gonzalez1,
- Marta Gonzalez1,
- Marta Gonzalez1,
- Marta Gonzalez1,
- Marta Gonzalez1,
- Marta Gonzalez1,
- Marta Gonzalez1
- 1Vall d'Hebron Research Institute
Protocol Citation: Marta Gonzalez, Marta Gonzalez, Marta Gonzalez, Marta Gonzalez, Marta Gonzalez, Marta Gonzalez, Marta Gonzalez 2022. Stereology-mediated cell count using StereoInvestigator. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4ekbgmk/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: September 13, 2022
Last Modified: September 13, 2022
Protocol Integer ID: 69934
Abstract
Protocol for cell counting using StereoInvestigator software
Materials
MBF Bioscience StereoInvestigator 11 (64 bits) Software (Micro Brightfield)
Set slide properly on the microscope and set new reference point
Click on Probes → Optical Fractionator Workflow → Start a new subject →
Enter cut thickness (5 µm (cut with microtome, 20 µm or 30 µm (cut with cryostat))
Enter interval according to the thickness: 17 if 5 µm, 6 if 20 µm or 4 if µm)
In Select Low Mag Lens, click 4X
Click on Next Step
Select the Contour of Interest(s)
In Select High Mag Lens select 100X
Click on Next Step
In Manually enter the average mounted thickness enter the corresponding one
Click on Next Step
In Define the Counting frame enter 50 x 50 µm
Click on Next Step
Enter 25 Percentage
Click on Display Changes
Enter 100x75 µm (if cut thickness is 5 µm) or 125x100 (if 20 µm or 30 µm)
Click on Display Changes
Click on Next Step
In Optical Disector Height enter 5 µm, 20 µm or 30 µm
Click on Next Step
Select your first section
Click on Yes
Select the desired region
Click on start counting
Focus on the tissue
Click on OK
Select a number of markers according to the desired cell types
Note
IMPORTANT 1: Count only the cells with visible nucleus within the square
IMPORTANT 2: If a cell touches a line of the counting frame: if it touches the green line, count the cell; if it touches a red line, do not count it.
Count every defined region from every set section
Click on Next Step
Click on Display Probe Run List
Click on Section Name → Click on Counter/Marker Name → Select all sections belonging to the same region
Click on View Results
Note
IMPORTANT: Verify that interval corresponds to the interval set in Step 3
Click on the different markers to see the number of cells counted and the estimated cell number
Click on CE Scheaffer to view the area if needed
Repeat steps 17 through 20.1 for each region