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Cell Counting of Dorsal Root Ganglia Fluorescence Microscopy Images

SOP Type: Data Analysis/Quantification
Date last revised: Oct 2022
Prepared by: Kate Salotto

CONTENTS
1. Revision Record
2. Purpose
3. Abbreviations & Definitions
4. Materials
5. Procedure
6. FAQs/Troubleshooting with Examples
7. Related Documents & References

1. Revision Record
Revision Date Prepared by Description of Change
1 October 2022 Kate Salotto Initial release

2. Purpose
To detail the process of extracting quantitative data from fluorescence microscopy images of
immunohistochemically stained mouse DRG samples through counting numbers of cells present and cells
infected by the experimental AAV.

3. Abbreviations & Definitions

 DRG – dorsal root ganglion. The collection of cell bodies from one vertebral level’s primary sensory
neurons. These neurons exhibit a pseudo-unipolar structure, so the cell body at one level of the spinal
cord.
 Cell body, neuronal soma (pl. somata/somas) – the part of the neuron that is generally rounded in
shape and contains cytoplasm and the nucleus.
 MAP2 – microtubule associated protein 2. This is the protein targeted by IHC for use as a control
stain/indicator of neuronal somata. A MAP2 stain fills the entire cytoplasm of the cell body and may
leave an unstained nucleus.
 EGFP – (enhanced) green fluorescent protein. Protein encoded by the AAV vector serving as a reporter
protein (its expression indicates successful integration of host and viral genetic material, and thus
successful infection of the cell). Excites at 488 nm.
 TdTomato – Red emission spectra reporter protein encoded by the AAV (alternative to EGFP).
 SGC – satellite glial cell (AKA satellite cell). A glial cell type located in the peripheral nervous system
that supports and communicates with the neuronal cell bodies in ways that are not fully understood at
this time. A SGC uses cytoplasmic projections to envelope the neuronal soma, thus controlling its local
environment. When stained, they appear as rings encircling or crescents hugging a cell body, often
with a small cell body of its own that exhibits nuclear clearing (see Section 7). All primary sensory
neuronal cell bodies are surrounded by at least one SGC. The greater the volume of the cell body, the
greater the number of surrounding SGCs and generally their volume.

4. Materials
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ImageJ or Fiji downloaded to a computer, template spreadsheet for documenting cell counts, images to be
quantified.

5. Procedure
General
The image data collected for this project are packaged in a single file as a series of images taken stepwise
at varying depths through the sample thickness. This set of images is called a Z-stack and each image is
called a slice. Additionally, each Z-stack contains 3 channels that correspond to the 3 excitation
wavelengths used to image every slice for the 3 IHC stains performed on the tissue samples.

Steps
1. Start the Fiji/ImageJ application and open an image file (File>Open…).
2. Open the Channels Tool

3. Select “Composite” from the dropdown options and then check/uncheck which channels you want to
see superimposed. I like to put the image in Composite display mode after opening the image because
it allows for quick toggling between overlapped and non-overlapped views in a single window while
still being able to scroll through the Z-stack. Further, when a composite image is initialized in the cell
counter plugin (described below), counting of different cell types can be done on across all three
channels at once, which is helpful for determining overlap/colocalization of fluorescent signal among
channels.
4. Open the Cell Counter Plugin (shown below).
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5. Click on the image stack to be counted, then click the “Initialize” button in the cell counter window.
6. Click a type on the left under the counter window. Use a different type for each of the cell types being
counted. This helps to keep track of counting MAP2 stained cells (Ch1), EGFP/tdTomato expressing
cells (Ch2), and satellite cells (Ch3 or Ch2 stained cells of satellite cell morphology).
7. Count for all image slices where the staining and cells are clear and free of artifact. See below on
performing Z-projection for speeding up this process.
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8. Transfer counts to data collection spreadsheet.

Z-Projection
A Z-project is when a stack of images is flattened into one output image. This is desirable because cells that
are split through multiple slices can be “flattened” and counted once in the final image instead of
recounting the same cells for every slice.
1. Open Z-project

2. Set range for slices to be included.

3. Select Max Intensity for projection type. Note: If the staining is very bright/saturated, then can try
Standard Deviation, which also works nicely. The Hong lab typically uses Max Intensity.
4. Open Cell counter plugin and count cells as described above.

See Example A.

Preprocessing Strategies
 Thresholding
 Masking

6. FAQs/Troubleshooting with Examples
# Problem encountered
1 Sample too thick/too many cell
layers -> Z-projection yields an
image too difficult to count.
2 Parts of the image are in focus
while others are out of focus or
have artifact from staining/slide
prep that obscure the final Z-
projected image, but stack is
otherwise a good candidate for
Z-projection.
See Example B below.
3 Staining is present but is faint or
has low contrast with high
background noise, making it
harder to count.
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What to try
1. Chunk the stack into smaller sub-stacks and perform a Z-
projection on each sub-stack. Count each Z-projection and
average results. Do as many sub-stacks as needed to get
clearer Z-projections to count.
2. Count slice-by-slice and average results.
Create image masks using ROIs (ROI Manager) to include in-
focus/unobstructed parts of the image for each slice of the stack
range you want to Z-project.
Draw a selection/ROI with the rectangle, polygon, or freehand
selection tool. Make selection a mask (Edit>Selection>Create
Mask) and right click in the new window generated to rename. Do
in order of stack.
Compile the mask images into a stack (Image>Stacks>Images to
Stack).
Split channels (Images>Color>Split Channels).
Remove the unwanted parts of the image by Process>Image
calculation
Select the image and the mask stack and choose AND for the
operation.
Perform max intensity Z-projection on the resulting stack for a
clearer Z-projection image that does not include artifact/out of
focus sections that obstruct the final image.
Can adjust brightness/contrast of picture to help distinguish while
counting. However, be careful not to introduce false positives or
bias the reading with an overly brightened image when using this.
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4 Not sure if fluorescence signal is If you are unsure, do not count it.
a true signal from a cell because
of…
 Faint/messy staining
 Artifact from imaging,
staining, or slide
preparation process
 High background
 Obscured view (such as by
axonal filaments from the
sample)
 High background noise
 Any other reason!
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Example A. Cell counting

Same Z-projection image with other cell types labeled:

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Green neuron cell body - label 1, white
Red virus infected cell body – label 2, pink
Ignore label 3
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Example B. Masking with Z-projection
Z-projection without using masks (lots of red staining artifact obscuring image center and left):

Z-projection after masking:

7. Related Documents & References

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Various satellite glial cell staining in the literature

*redacted*
Figure 1. Vimentin stain (red, B and E) of DRG in WT mice (sciatic n. transection). Below are B and C
shown larger.

*redacted*
Figure 2. GFP expression by AAV infected neurons and SGC (arrows) in (A).

*redacted*
Figure 3. Staining of SGC for glutamine synthetase. Arrows point to 2 examples of satellite cell bodies.
Exhibits non-staining nucleus with staining cytoplasm and projections that surround the neuronal
somata.
*redacted*
Figure 4. GFAP staining of A) SGC and B) (inflammation) activated SGC.