I came across the use of scoring in immunohistochemistry. Can someone please help me understand how I perform this scoring in tissue section for my DAB stained sections?
There are two methods for IHC scoring 1) Automated software based and 2) Manual method. The automated though faster has many inherent problems and personally I do not prefer. I use the manual method. For this have a cursory examination of your staining and set up a score from 0-5. o being no staining to 5 being maximum staining. 1,2,3,4 will have to be decided arbitrarily.
Now at 100X magnification in each field count the number of cells with the different staining intensities. Count 10 random fields in three technical replicates to reach a minimum count of 500 or more. Use the following formula to generate the H score. H Score = summation (1+i)pi where i is the intensity score and pi is the percent of the cells with that intensity.
e.g. H score = (1+0)20 + (1+1)5 +(1+2)10 + (1+3) 35 + (1+4) 20 +(1+5)10 = 360.
Generate scores for all your biological replicates. Generate mean SD for the score and apply t test for statistical significance.
Hope this helps.
Best luck
Article Regulation of homeobox A10 expression in the primate endomet...
There are two methods for IHC scoring 1) Automated software based and 2) Manual method. The automated though faster has many inherent problems and personally I do not prefer. I use the manual method. For this have a cursory examination of your staining and set up a score from 0-5. o being no staining to 5 being maximum staining. 1,2,3,4 will have to be decided arbitrarily.
Now at 100X magnification in each field count the number of cells with the different staining intensities. Count 10 random fields in three technical replicates to reach a minimum count of 500 or more. Use the following formula to generate the H score. H Score = summation (1+i)pi where i is the intensity score and pi is the percent of the cells with that intensity.
e.g. H score = (1+0)20 + (1+1)5 +(1+2)10 + (1+3) 35 + (1+4) 20 +(1+5)10 = 360.
Generate scores for all your biological replicates. Generate mean SD for the score and apply t test for statistical significance.
Hope this helps.
Best luck
Article Regulation of homeobox A10 expression in the primate endomet...
For the H-score for ER and PR in Breast Pathology the easiest way is :
The H-score is a method of assessing the extent of nuclear immunoreactivity, applicable to steroid receptors. The score is obtained by the formula:
3 x percentage of strongly staining nuclei + 2 x percentage of moderately staining nuclei + percentage of weakly staining nuclei, giving a range of 0 to 300.
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