I need to known of a non-biased and objective quantification method or software to estimate or count the number of immuno-reactive cells across multiple brain areas and for a number of neurochemical markers (DA, NE, 5-HT, GABA, and GLU) of developmental toxicity.
We use the MicroBrightField Stereoinvestgator according to the method described by Gundersen.
http://www.ncbi.nlm.nih.gov/pubmed/3288247
there are several books on the topic. I agree that the Gundersen explanation is very straightforward. I use the book, Unbiased Stereology: Three-Dimensional Measurement in Microscopy by Howard and Reed to help my students understand the concepts - it has some very nice illustrative problems to solve for practice. Mark West has also written books, and occasionally hosts a stereology workshop.
We use the MicroBrightField Stereoinvestgator as Björn Spitau. We have used it to quantify immunohistochemistry in nervous tissue of ANTI-HTR2A and with protein expression in pituitary gland with good results. It is a much reliable and objective system that is widely accepted in scientific publications. The only problem is that you need a computerized microscope to apply it.
You can use the microbrightfield software with confocal image stacks - eliminating the need for a computerized microscope
By the expertise of L. Portiansky. We, after a deconvolution processes, analysed by using algorithms
developed with an image analysis program (ImagePro Plus, v6.3, Media Cybernetics, USA). It works very well!! You can ask about it to Portiansk: [email protected]
Cell Tissue Res. 2010 Jul;341(1):181-95. doi: 10.1007/s00441-010-0990-8. Epub 2010 Jun 8.
Neuroanatomical distribution of angiotensin-II-like neuropeptide within the central nervous system of the crab Chasmagnathus; physiological changes triggered by water deprivation.
Frenkel L, Dimant B, Portiansky EL, Imboden H, Maldonado H, Delorenzi A.
http://www.ncbi.nlm.nih.gov/pubmed/20526894
We use MicroBrightField Stereoinvestgator for unbiased stereology. This can done either online using a microscope running the SteroInvestigator as well as off-line by using z-stacks that were obtained with another microscope.
Concerning the use of antibodies: Thicker sections are better than thinner sections (in case of a stereological approach). However, it should be tested whether the antibodies are able to fully penetrate the thicker sections (in the worst case the cells located in the middle of the sections will not be stained). Thus, I would recommend to run some preliminary experiments to test this.
We'll do - thanks to all for your expert suggestions. We'll have a "dry" run so to speak to determine the optimal thickness - considering 40 um of rat brain - and free floating staining to ensure the antibodies fully penetrate the sections. We'll have to think harder about stereology about we'll get there.
Hi, I did some work on the the Neuropile components estimation in primate brains in a thesis submitted to The University of the |witwatersrand , Johannesburg. You should have access to it and check the methids i employed, if they will be of help to you.
Friend of mine did it in lung sections, well published, look for H. Fehrenbach as AU
If you do not have the StereoInvestigator setup, I would recommend starting by reading Rakic's papers on unbiased stereology. A number of people have applied similar principles to freeware such as FiJi, and by utilising confocal images. Application of the abercrombie method has also been applied to brightfield images but there is debate about its accuracy.
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