Although this was mentioned in the previous answers - you CANNOT quantify the DAB based staining by measuring staining intensity. DAB does not fulfill the Beer-Lambert law and is not stoichiometric, therefore the staining intensity is not related to the number of antigens (see e.g. discussion there: http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html). You can quantify (semi-quantify) DAB staining by e.g. counting the number of cells using stereology, or by measuring the area covered by the staining.

I considered as qualitative method rather than quantitative. Let us hear from experts

Actually, all immunohistochemistry whether DAB or immunoflourescence are not purely quantitative. They are semi-quantitative techniques. Also, the purpose of immunohistochemistry is not mainly quantification, but intracellular and tissue localization of the protein of interest. You may not be able to accurately quantify the protein, but the way it is normally done is by histometry or subjective scoring. Histometry calculates the intensity of staining in the number of cells taking up the stain. Softwares like qWin from Leica etc are very user-friendly for this purpose (it is licensed!). Subjective scoring is a method in which number of immunostained cells per 200 cells in field is calculated and intensity scores from 0 - 5 are given in which 0 corresponds to no visible stain and 5 corresponds to maximum staining. Comments then, additionally, can be made on the localization (nuclear, cytoplasmic, membrane bound) etc and also the cell type that takes up the stain (stroma, epithelial lining, glands, endothelium etc.). These are, to my opinion, the information that can be extracted from IHC.

We quantify immunohistochemistry data with DAB, and also tissue immunofluorescence. Nevertheless it is a semi-quantitative technique as Ms. Anupa mentioned above. If you are looking for a tissue quantitative technique, I would suggest flow cytometry on newly collected tissues. You can quantify and compare protein expression in a IHC (knowing that it is semi-quantitative, similar to a immunoblot); and compare protein localization.

Thank you everyone for your input. I am curious to know from Edwardo what method you use to semi-quantify immunohistochemistry data with DAB?

Also, does anyone know of an ImageJ plugin that can semi-quantify DAB immunohistochemistry data?

Dear Amjad,

I work with Image Pro Plus to quantify DAB staining. In ImageJ I use only for immunofluorescence, which quantifies gray gradients. In Image Pro Plus, there is a way in which you can quantify the areas in which DAB stained the cell by selecting a color (or mean colors), and these colors are then area-selected in the whole picture and quantified.

Although this was mentioned in the previous answers - you CANNOT quantify the DAB based staining by measuring staining intensity. DAB does not fulfill the Beer-Lambert law and is not stoichiometric, therefore the staining intensity is not related to the number of antigens (see e.g. discussion there: http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html). You can quantify (semi-quantify) DAB staining by e.g. counting the number of cells using stereology, or by measuring the area covered by the staining.

I would refer to the protocol exchange published online on the nature website that explains how the reciprocal intensity quantification of chromogen in IHC works.

https://www.nature.com/protocolexchange/protocols/2931

Thank you Dariusz and Eduardo. It seems that, at least for my study, the way to semi-quantitate DAB IHC is by measuring areas and comparing between groups. Is it fair to run statical analysis on area data or would I be limited to qualitative comments about the differences in areas between groups?

Dear Amjad - measuring the areas is an option in your case, but it is difficult to say anything more without knowing your data set. For proper analysis you will need to measure the stained area in series of sections, the sections for different groups should be processed exactly the same (same batch of Ab, same procedure (optimally the sections from all the groups should be processed together to avoid small, but possible, differences in the protocol), then the photographs for analysis should be taken using the same settings for the microscope and attached camera. Then you can measure the stained areas using image analysis program - e.g. ImageJ (the best using the same setting for threshold, what not always is possible due to e.g. uneven staining pattern or high background - in such case you may set threshold manually, but you must ensure that only the stained cells will be included in analysis (therefore, I would call such analysis semi-quantitative)), if your section number(animal number) is sufficient you can calculate the statistics, if not you can present stained areas comparison with qualitative description.

To state in more simple terms to what Dariusz had to say, the DAB reaction is very time-sensitive (and goes quite fast - thus is hard to control) and deposits the substrate around the site of the HRP (not on the HRP). Additionally, the HRP activity can vary between lots of the HRP conjugate. You cannot precisely control the amount of substrate that is deposited. Thus, you cannot precisely measure the degree of labeling. Because of this, any quantitation you attempt will have a high degree of variability. Therefore, it is more qualitative and only semi-quantitative. Can you quantify? Yes, but you have to accept that high degree of variability and have a high number of replicates and very precisely timed labeling using the exact same reagents.