Computational protocol: Neonatal Administration of Thimerosal Causes Persistent Changes in Mu Opioid Receptors in the Rat Brain

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Protocol publication

[…] The specimens were stained using Rabbit Anti-Mu Opioid Receptor Monoclonal Antibody Clone EP1470Y (1:100) (from Abcam, USA) and R.T.U. Vectastain Kit (from Vector Laboratories, USA), and then treated with: diaminobenzidine and hydrogen peroxide solution (1 ml DAB + 1 ml TRIS–EDTA solution + 1.6 μl 30% hydrogen peroxide; 3 min), ethanol, xylene, finally immersed in Histofluid (Marienfeld) using cover glasses. The microphotographs of the brain sections were made with the Olympus BX41 microscope and Olympus DP25 digital camera. The images were saved in TIFF format. For each experiment microphotographs were made with the same light level for all sections.To evaluate MOR density, the following sections were used for analysis: (1) sections showing the periaqueductal gray (PAG) (Bregma -6.36) included regions: the dorsomedial periaqueductal gray (DMPAG) and the lateral periaqueductal gray (LPAG); (2) sections showing the caudate putamen (Bregma -1.20) were analyzed in the North-East quadrant; (3) sections showing the hippocampus (Bregma -3.60) were used to analyze the dentate gyrus. The position of the regions was determined according to the rat brain atlas []. For analysis of receptor density, microphotographs of each region were taken with a magnification of 400× (area of 35452.23 μm2). The MOR density was counted as an area fraction in the analyzed region, with ImageJ 1,41o software and used for statistical analysis. The area fraction was automatically counted with a “threshold” function of the ImageJ program, which marks all the pixels of chosen gray value and counts all the groups of marked pixels (stained receptors) within the selected area []. [...] The STATISTICA software package for Windows (StatSoft, Tulsa, OK, USA) was used to analyze all data. An analysis of variance (ANOVA) was used to compare experimental groups. An LSD test was employed for individual post-hoc comparisons. Probability (P) levels less than 0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus