Bitan:MicrogliaIHC

Bring section under focus in StereoInvestigator

Acquisition → Acquire Image

Click on the image, but not on the section

Image → Add Scalebar

Set to 100 microns

Apply Scalebar

To get to the next section revert back to Live Image and Select New Data File

Open Image in ImageJ

Open a second image

Both Images: Image → Type → 8-bit

One Image: Analyze → Set Scale 70 pixels → 100 μm Process → Subtract Background 50 pixels rolling ball radius Check Light Background Use color picker to pick a color closest to white Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section Image → Adjust → Brightness and Contrast Adjust min/max bars until microglia are clearly contrasting the background Compare with other 8 bit image Image → Adjust → Threshould Adjust Threshold value until microglia are selected and as much background is removed as possible Use zoom tool to compare with 8-bit image Analyze→ Analyze Particles Select Summarize and DIsplay Measurement Mark down: Count, Total Area, and Average Size