Eclips e200 microscope

All 124 male mice from (B6.PWD-Chr X×B6)F1×PWD cross and 71 male progeny from (B6.PWD-Chr X×B6)F1x B6 cross were genotyped using SSLP Chr X markers listed in Table S6. Genomic DNA from mouse tails and spleens was prepared by the HotSHOT method [64] (link) followed by phenol-chloroform cleaning [65] (link). For genotyping B6.PWD-Chr X# partial consomics and 84 males of B6×(PWD×STUS) test-cross we used MegaMUGA high-density genotyping array carrying 77,000 markers on Illumina Infinium Platform (http://csbio.unc.edu/CCstatus/index.py).
Eight weeks old male progeny of the crosses were phenotyped for testes weight (TW), sperm count (SC) and percentage of abnormal spermatozoa as described [33] (link). The B6×(PWD×STUS) males were phenotyped at 60 days of age as described [44] (link). For histological analysis the paraffin-embedded testicular sections were stained with periodic acid Schiff and hematoxylin-eosin and observed using a Nikon Eclipse 200 microscope. The Penguin 150CL CCD color camera (Pixera) was used to capture photographs, which were processed using Adobe Photoshop (Adobe Systems).

Liver samples were removed from the animals of the in vivo experiments after collection of blood and were fixed overnight in 10 % buffered formalin. The samples were subjected to dehydration and then embedded in paraffin blocks. Thick sections (4 μm) of the paraffin embedded livers were cut in a microtome and then dewaxed in xylene, rehydrated in a series of different grades of alcohol and then washed with distilled water for 5 min. Subsequently, the sections were stained with haematoxylin for 40 s and counterstained with eosin for 20 s. The sections were dehydrated in graded alcohol series and washed in xylene. The slides were observed (100× and 400×) for signs hepatic injury using Nikon ECLIPS E200 microscope.

Livers were removed from the experimental mice, cut into small pieces and fixed in 10% formaldehyde solution for overnight followed by dehydration. Dehydrated tissues were embedded in paraffin. 4 μm sections were cut using microtome. Then liver sections were dewaxed in xylene, rehydrated in a series of different grades of alcohol and then washed with distilled water for 5 min. The liver sections were stained with basic stain haematoxylin for 40 sec and counterstained with acidic stain eosin for 20 sec. After proper staining the slides were observed (100X and 400X) using Nikon ECLIPS E200 microscope to identify the damages like necrosis, portal inflammation, vascular congestion, fatty infiltration, vacuolar degeneration, leukocyte infiltration, loss of structure of hepatic nodules and so forth [29 ,30 ]. Fibrosis was also observed in the CCl₄ intoxicated group.