immunohistochemistry quantification - (Dec/07/2006 )
recently I start doing immunohistochemistry for the tissue slides. I am wondering if anybody could help me to quantify it? which method is easier and software? any information will be appreciated.
No one can help you quantify because it isn't quantitative.
Even if there is software available for quantitation (I'm not sure if it exist)
it is simply not feasible to quantify a precipitation that acuurs on a slide that is dependent on how
long you let your reaction run. ie. Short reaction time = light staining, long reaction time = strong staining.
All you can do is a negative IgG control and say if it is there or not and maybe speculate on the relative levels. Anything more than this would be simply bogus.
Mikew is correct IHC is not quantitative and I agree that any relative quantitation you try to do is not very accurate, especially if you are comparing different sections, but you can compare the relative levels of staining.
The most accurate way would be to compare staining in two different cell types in the same section. I have also done a very rough quantitation using two different sections that were on the same slide and treated identically including the same staining time.
I don't know if any software is available, but I just used photoshop. I took pictures of each section under the same magnification and light conditions. I then selected the cell of interest in Photoshop and used the histogram function to get the mean intensity of the pixels. Darker staining in IHC equals higher but in Photoshop it measures the lighter pixels as higher, so you can either invert the picture first or just know that a lower mean equals higher staining.
You also need to subtract the background, which is complicated. I think the best way is to choose a cell in the same section (not your control section) that does not express the protein, so you can account for non-specific staining.
I selected 50 cells at random from each section and took the mean of all 50. And was able to see that the staining in sections from my transgenic mouse was higher than the wild type and it was statistically significant.
many thanks to Zona Pellucida and mikew.
if you do CLSM you find appropriate confocal software with support of your aim; you can also use any quantification program if you can define a ROI