Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software.

@article{citeulike:2784528, abstract = {This paper describes simple procedures to process digital images in quantitative immunofluorescence microscopy. Monoclonal antibodies directed against the sarcoplasmic myosin heavy chain isoforms and against laminin, located on the basement membranes, were applied to sections of human skeletal muscle. The localisation and staining intensity of a fluorescent secondary antibody were recorded using an indirect histochemical method. The digitised images were pre-processed and the luminosities of appropriate structures were determined using existing tools in the widely used image processing software Photoshop from Adobe. Procedures to obtain a quantitative measure for the specific fluorescence signal (the background corrected fluorescence in the object) were developed. In addition, antibody binding to individual cells could be quantified whether these cells are well separated or not. The relation between the specific fluorescence signal and the dilution factor of the primary antibody could be measured to determine a suitable concentration of the antibody for incubation of the sections. The potential fading of the fluorescence signal with time and prolonged exposure to light from the microscope was explored and analysed. With the tools described in the present report it is thus possible also to optimize the topical immunohistochemical protocol in order to quantify the fluorescence signal.}, address = {Department of Oral Medicine, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, 20 Nørre All\'{e}, DK 2200 Copenhagen, Denmark. sk@odont.ku.dk}, author = {Kirkeby, S. and Thomsen, C. E. }, citeulike-article-id = {2784528}, doi = {10.1016/j.jim.2005.04.006}, issn = {0022-1759}, journal = {Journal of immunological methods}, month = {June}, number = {1-2}, pages = {102--113}, posted-at = {2008-05-11 16:03:17}, priority = {2}, title = {Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software.}, url = {http://dx.doi.org/10.1016/j.jim.2005.04.006}, volume = {301}, year = {2005} }

TY - JOUR ID - citeulike:2784528 TI - Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software. JF - Journal of immunological methods VL - 301 IS - 1-2 SP - 102 EP - 113 AD - Department of Oral Medicine, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, 20 Nørre Allé, DK 2200 Copenhagen, Denmark. sk@odont.ku.dk SN - 0022-1759 N2 - This paper describes simple procedures to process digital images in quantitative immunofluorescence microscopy. Monoclonal antibodies directed against the sarcoplasmic myosin heavy chain isoforms and against laminin, located on the basement membranes, were applied to sections of human skeletal muscle. The localisation and staining intensity of a fluorescent secondary antibody were recorded using an indirect histochemical method. The digitised images were pre-processed and the luminosities of appropriate structures were determined using existing tools in the widely used image processing software Photoshop from Adobe. Procedures to obtain a quantitative measure for the specific fluorescence signal (the background corrected fluorescence in the object) were developed. In addition, antibody binding to individual cells could be quantified whether these cells are well separated or not. The relation between the specific fluorescence signal and the dilution factor of the primary antibody could be measured to determine a suitable concentration of the antibody for incubation of the sections. The potential fading of the fluorescence signal with time and prolonged exposure to light from the microscope was explored and analysed. With the tools described in the present report it is thus possible also to optimize the topical immunohistochemical protocol in order to quantify the fluorescence signal. AU - Kirkeby, S AU - Thomsen, CE PY - 2005/06// UR - http://dx.doi.org/10.1016/j.jim.2005.04.006 ER -