How to Quantitatively Analyze the Results of Immunohistochemistry and Western Blot
Quantitative Analysis of Immunohistochemistry
1. Image Acquisition
Use a microscope to capture images of stained tissue sections, ensuring that all imaging parameters (e.g., exposure time, light source intensity) are maintained consistently across all samples.
2. Image Analysis
Open the acquired images using image analysis software such as ImageJ.
3. Threshold Setting
Set a threshold based on background levels and staining intensity to accurately differentiate stained regions from the background.
4. Measurement
Measure the area and average intensity within the thresholded regions to obtain quantitative values for each region.
5. Normalization
Normalize the results using data from the control group or by comparing them to other markers.
Quantitative Analysis of Western Blot
1. Image Acquisition
Capture blot images using X-ray film or a digital imaging system.
2. Select Appropriate Software
Use image analysis software such as ImageJ or Quantity One for quantification.
3. Background Subtraction
Select a region of the blot without visible protein bands to define the background and subtract it using the software.
4. Band Quantification
Use the software tools to delineate each band and measure its optical density.
5. Normalization
Normalize the intensity of the target protein bands using internal loading controls such as β-Actin or GAPDH.
6. Data Analysis
Perform further data analysis using graphing or statistical software.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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