The Threshold and Calculate Average Intensity with Brush Tools macro is customized for immunohistochemistry image analysis done in the Ogle Lab. It is especially suited for stained tissue sections that have large areas of background interspersed between areas of signal.
- Drag in all fluorescence images into ImageJ/Fiji.
- Run the command: Plugins > LOCI >Threshold and Calculate Average Intensity with Brush Tools.
- Optionally drag in any Brightfield images when prompted. Brightfield images can guide region of interest highlighting, but are not analyzed for intensity.
- User sets threshold to include only desired pixels to be analyzed. It is recommended to choose the low level above the scattering from the glass, below the tissue fluorescence. Usually the high level should be set to the max. DO NOT click any of the buttons in the threshold window, but instead click OK in the prompt window.
- nbsp; Exclusion tool: The user can use the brush tool along with SHIFT + CLICK to outline areas that should not be included in the calculation (areas that made it through the threshold).
- Inclusion tool: The user can use the brush tool along with SHIFT + CLICK to outline areas that should not be included in the calculation.
- The algorithm calculates the average intensity of the red and included areas and presents it in the results window.