Computational protocol: Immunohistochemical detection of angiotensin AT 1 and AT 2 receptors in prostate cancer

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Protocol publication

[…] The expression of both receptor proteins was evaluated quantitatively using the image analysis methods described previously []. The software tool used for immunoquantification was ImageJ ver 1.42p developed at the US National Institutes of Health ( The microscopic images were gathered as RGB digital images (24 bits per pixel) using an Olympus camera (type C7070) with a linear resolution of 5.6 pixels per 1 micrometer under original magnification approx. 400x, and under the same lighting parameters (the automation of camera was switched off). For the analysis, the images with carcinomatous tubules (or solid infiltration in the case Gleason 5 cancers), non-neoplastic tubules, and/ or capillary vessels were recorded. All these structures were lying in the vicinity, i.e. were visible in one medium power field (magnification 100 x). The red cells that were not superimposed in the vessel acted as the reference densitometric value for determination of the residual peroxidase activity, which manifested as a trace expression of DAB. In each investigated case, the image analysis was performed in 5-6 fields, each measuring 0.232 mm2. The image analysis procedure consists in conversion of tubule crosssections image, in order that the resulting image for densitometric measurements was the mask of „pure” cytoplasmatic outlines with DAB staining, without tubular lumina, intercellular space or shapes of nuclei. For this purpose the color masks in RGB images and color filters inserted into the optic were used. The extraction of brown stained cytoplasm was performed with blue filter and of brown stained cytoplasm was performed with blue filter and blue mask (the supplemented color for DAB dye), and the extraction of blue stained nuclei was performed with orange filter and red mask (the supplemented color for hematoxylin dye). The upper cut-off luminance level of DAB stained cytoplasm (for extraction of lumina or intercellular space) was fixed according to the reference densitometric value of red cells. Small manual corrections sometimes were needed. The shapes of nuclei and the shapes of lumina and intercellular spaces were cut from the image of DAB staining. The densitometric values of immunohistochemical expression of AT1 or AT2 receptor proteins were measured in extracted areas of the originally recorded image. The integrated optical density of red cells (i.e. mean graylevel of one pixel) were divided by the integrated optical density of the measured structure. The maximal brightness (i.e. lack of DAB staining) was 255 graylevels, the minimal was 0 (theoretically maximal intensive DAB staining). Thus, a higher relative value indicates a more intense DAB expression. The results are presented as relative values according to the original primary histological grading in every field, not according to the total sum for every case (i.e. to the total Gleason score).The statistic analysis was performed using Statistica ver. 5.0 software. After the F-Snedecor test for variance equality, the appropriate double-sided t-Student test was used for estimating the statistic significance of differences of densitometric values of various types of glands (p <0.05). Moreover, the Pearson correlation coefficient r was calculated. The correlation between densitometric values of glands in the same areas stained with AT1 and AT2 antibodies was analyzed. The steps of the image analysis are presented below (–). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Prostatic Neoplasms
Chemicals Biotin, Eosine Yellowish-(YS), Hematoxylin, Paraffin