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Author Topic: DAB Staining Intensity for Quantification?  (Read 12769 times)

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Offline rockie

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DAB Staining Intensity for Quantification?
« on: December 04, 2009, 04:46:59 PM »
Hi, I have a question about DAB staining. In addition to quantifying the number of positive cells identified by DAB staining, can I also look at the intensity of the staining as a way to determine expression level? ie. light brown versus a dark brown staining.

Note, I have a positive control on each slide and their intensity is the same, but there is a big difference in the intensity of the signal that I am looking at. ie. one very strong dark brown color and the other is light brown. All samples are processed at the same time.

Thanks in advance to anyone who can help.

DAB Staining Intensity for Quantification?
« on: December 04, 2009, 04:46:59 PM »

Offline gula

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Re: DAB Staining Intensity for Quantification?
« Reply #1 on: December 05, 2009, 12:55:44 PM »
Eg. for her2 testing we use a semiquantitative testing with 0,1+,2+,3+. So I think, there is a direct relation between antigen-amount and staining intensity. But I doubt, that you can make an exact quantification by comparison of tissue-slides.
As seen with her2 there are many components, that can influence the result beginning with fixation, antibody-titer, antigen-retrieval etc. I think that's the big difference between tissue and other specimens like serum-parameter and ELISA testing.
gula

Offline Babybio

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Re: DAB Staining Intensity for Quantification?
« Reply #2 on: February 15, 2010, 12:38:31 AM »
Take a look at the paper below, as it has a very useful method and algorithm that will help.  Also, you can use Image J to look at specific spatial expression patterns

Sia Y, Bourne JA. (2008) Neuroscience. 156(1):118-28.http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0F-4SX3P39-3&_user=542840&_coverDate=09%2F22%2F2008&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000027659&_version=1&_urlVersion=0&_userid=542840&md5=8f14c324faf08e90cb2a74064120b1cd
The rat temporal association cortical area 2 (Te2) comprises two subdivisions that are visually responsive and develop independently.

In this study, we have used the expression of non-phosphorylated neurofilament (NNF), a protein that exhibits differential areal and laminar neuronal patterning, to assess the chemoarchitectural organization of the rat temporal association cortex (Te). Since expression of NNF is associated with the latter stages of neuronal development, this enabled us to profile the hierarchical development of this region of the cortex. We also examined the expression of the protein Fos, the product of the immediate-early gene cFos, as a neuronal activity marker to determine which areas within this region are visually responsive. Our findings reveal the existence of two previously undescribed subdivisions within the dorsal and ventral domains of the rat temporal association cortical area 2 (Te2) which we have termed Te2d and Te2v, respectively. We also demonstrated the early maturation of the caudal region of Te2d while preceding the primary visual cortex. Within this region of the cortex, the Fos protein indicates that both subdivisions are visually responsive.


Re: DAB Staining Intensity for Quantification?
« Reply #2 on: February 15, 2010, 12:38:31 AM »