Image J Protocol - David Rempel：Image J协议-戴维Rempel.ppt

BE1151 1. Open Image J and, in it, open the .TIF file to be analyzed - in this case, “ ”. BE1152 1b. Optional ? If the image is not already an 8-bit .TIF, go to Image -> Type and select 8-bit . ? If the image is not dark enough to analyze properly, go to Image -> Adjust -> Brightness/Contrast and adjust the slider bars until the bands on the image are visible. The pixel values will not be changed, just the view. BE1153 2. In the main menu, select Analyze > Calibrat e. BE1154 3. Select the Uncalibrated OD option from the Function: dropdown box, then click O K. BE1155 4. A logarithmic plot should appear; move it to the side, but do not close the window. BE1156 5. Go back to your .TIF image and select the Rectangle Tool from your toolbox. Box all of the bands of interest across all of the lanes and hit Crtl-H to bring up a histogram. BE1157 6. Carefully outline the first lane with the same tool. Go to Analyze > Gels > Select First Lan e. BE1158 7. Drag the rectangle that you used for the first lane over to the next, and mark it with Analyze > Gels > Mark Next Lane. Don ’ t draw a new rectangle! You want every rectangle to have the same area. Repeat until all the lanes have been marked. BE1159 8. Click on Analyze > Gels > Plot Lane s. You ’ ll end up with an image file with plots for each of the lanes that you marked. The plot on top is the first lane that you marked. Use Image > Rotate 90 Degrees Right (or Left) and/or Image > Zoom > Out so you can see and work with all of the plots. BE115 10 (OPTIONAL) If you have more than one band per lane, you ’ ll end up with a plot for a single lane that has multiple peaks (one for each band). You ’ ll need to separate them into individual peaks with the Line Tool as demonstrated below on another image .