D.) prior to immunohistochemical analysis.Sertoli cell stereologySertoli cell numbers were
D.) prior to immunohistochemical analysis.Sertoli cell stereologySertoli cell numbers were determined using a stereological method based on that reported by Paul and EPZ004777 web colleagues in 2005 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27906190 [34]. In brief, 5 m sections were stained with haematoxylin Z (Cellpath plc, Hemel Hampstead, UK) and, from each section, 40 images were taken (10 from each pole) using a magnification of x 630. The software ImageAndrade et al. Journal of Negative Results in BioMedicine 2013, 12:2 http://www.jnrbm.com/content/12/1/Page 7 ofPro Plus (Media Cyberbnetics, Wokingham, Berkshire) was used to generate a grid consisting of 432 evenly distributed points. This was superimposed over each digital image and the number of intersections on the grid overlying a Sertoli cell nucleus was counted using a manual tag system. The `total point count’ for Sertoli cells obtained was then expressed as a percentage of the maximum count possible (40 images x 432 possible intersections = 17,280) to give the `total point count percent’. This value was used to calculate the weight of testis made up of Sertoli cells and subsequently converted to volume occupied by Sertoli cells (absolute volume: AV) assuming 1 g of testis = 1 cm3 [34]. Image pro plus (Media Cyberbnetics) was then used to measure the diameter of 2 Sertoli cell nuclei per image. Since Sertoli cell nuclei are PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28154141 not spherical, the program took the average length of several diameters. The mean nuclear volume (MNV) of each Sertoli cell was calculated from the diameter as 4/3r3. The total number of Sertoli cells per testis was then calculated AV(m3)/MNV(m3)] and adjusted for testis weight so that values were expressed as number of Sertoli cells per gram of testis.Quantification of immunostainingThe immunohistochemistry results for Ki67, Bax and Mcl-1 were quantified by computer-aided image analysis. The image analysis system comprised an Olympus Corp. microscope (x 20 objective; New Hyde Park, NY) and Hamamatsu digital camera (Hamamatsu, Bridgewater, NJ) connected to a computer running Image-Pro Plus software (Media Cyberbnetics). Quantification by image analysis was conducted over six randomly selected fields of view, after which the mean and standard error had stabilised. The total area of positively stained cells (brown colour) was measured and expressed as a percentage of the total cellular area. Immuno-histochemical staining intensity and area for c-kit were measured visually and semi-quantitatively using an arbitrary four point scale. A score of 3 indicated intense staining, 2: moderate staining, 1: some positive staining and 0 indicated no staining. Cord and interstitial areas were assessed independently and all analyses were carried out by the same operator.Statistical analysisHistology and immunocytochemistryAll tissue sections were de-waxed in Histoclear (National Diagnostics, Hessel, Hull, UK), re-hydrated through a graded ethanol series (100 , 95 , 70 ) and washed in Tris-buffered saline (TBS; 0.1 M Tris Cl; pH 7.6; 0.85 NaCl) for 2 x 5 min. Antigen retrieval procedures were necessary for exposure of all epitopes and this was achieved by microwaving sections in 0.01 M citrate buffer (pH 6.0) on full power (100 , 700 Watt) for 3 ?5 min. Sections for both studies were placed in a DAKO Autostainer (DakoCytomation, Ely, UK) and incubated with the appropriate primary antibody for 30 minutes. Primary antibodies were as follows: (a) monoclonal mouse anti human Ki67 at a 1:100 dilution (Clone MIB-1: DakoCytomation, Ely, UK).
