Bitan:MicrogliaIHC: Difference between revisions
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(Both StereoInvestigator and ImageJ Protocol to find the area of section stained) |
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Bring section under focus in StereoInvestigator | == Bring section under focus in StereoInvestigator == | ||
Acquisition → Acquire Image | Acquisition → Acquire Image | ||
Click on the image, but not on the section | |||
*Click on the image, but not on the section | |||
Image → Add Scalebar | Image → Add Scalebar | ||
Set to 100 microns | |||
Apply Scalebar | *Set to 100 microns | ||
*Apply Scalebar | |||
To get to the next section revert back to Live Image and Select New Data File | To get to the next section revert back to Live Image and Select New Data File | ||
Open Image in ImageJ | |||
== Open Image in ImageJ == | |||
Open a second image | Open a second image | ||
Both Images: | Both Images: | ||
Image → Type → 8-bit | |||
*Image → Type → 8-bit | |||
One Image: | One Image: | ||
Analyze → Set Scale | |||
70 pixels → 100 μm | *Analyze → Set Scale | ||
Process → Subtract Background | |||
50 pixels rolling ball radius | :* 70 pixels → 100 μm | ||
Check Light Background | |||
Use color picker to pick a color closest to white | *Process → Subtract Background | ||
Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section | |||
Image → Adjust → Brightness and Contrast | :*50 pixels rolling ball radius | ||
Adjust min/max bars until microglia are clearly contrasting the background | |||
Compare with other 8 bit image | :*Check Light Background | ||
Image → Adjust → | |||
Adjust Threshold value until microglia are selected and as much background is removed as possible | *Use color picker to pick a color closest to white | ||
Use zoom tool to compare with 8-bit image | |||
Analyze→ Analyze Particles | *Use pencil to whiteout all dark areas not part of the spinal section or are “spots” on the section | ||
Select Summarize and DIsplay Measurement | |||
Mark down: Count, Total Area, and Average Size | *Image → Adjust → Brightness and Contrast | ||
:*Adjust min/max bars until microglia are clearly contrasting the background | |||
:*Compare with other 8 bit image | |||
*Image → Adjust → Threshold | |||
:*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 | |||
Latest revision as of 04:33, 5 September 2015
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 → Threshold
- 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
