Eclipse e800

One uterine horn per mouse was fixed overnight in 10% neutral buffered formalin, processed in increasing concentrations of ethanol, and paraffin embedded. Tissues sections (5μm) were processed for histological analysis. Sections were stained with Hematoxylin and Eosin and analyzed by two pathologists blinded to the experimental exposures. For immunohistochemistry, sections were incubated with antibody specific to phospho-histone H3 (pHH3), alpha smooth muscle actin (αSMA) and cytokeratin 8 (CK8) (S1 Table), then with goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin. Representative images were taken on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. We used primary antibody against pHH3 for immunofluorescence (S1 Table) followed by goat Alexa Fluor 488—conjugated secondary antibody (Molecular Probes, 1:1000). Sections were counterstained with DAPI imaged by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software).

Prefer solution (Anatech Ltd, Battle Creek, MI) was used to fix the mouse eyes for 15 min at room temperature, followed by 70% ethanol overnight. The tissue was paraffin-embedded, and 5-μm-thick sections were cut and mounted onto slides. These sections were subjected to immunohistochemistry. A Nikon Eclipse E800 microscope and Olympus MVX10 macroscopic imaging system for high resolution equipped with a digital camera was used to examine the antibody-labeled complexes. Metamorph (Universal Imaging, West Chester, PA) image analysis software was used to capture images under identical microscope and camera settings.

Cells on coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with ice-cold 100% methanol (5 min) at −20℃, or 0.2% Triton X-100 (for LAMP1 and Rab7 staining) at room temperature, followed by blocking with 5% normal goat serum in phosphate-buffered saline (PBS), for 1 hr (Fortun et al., 2007 (link)). After an overnight incubation with primary antibodies (Table 1) at 4℃, samples were treated with Alexa Fluor 594 (red) or Alexa Fluor 488 (green) anti-rabbit, anti-mouse, or anti-rat secondary antibodies (Molecular Probes, Eugene, OR) for 2 hr at room temperature. Nuclei were visualized with Hoechst dye. Samples without primary antibodies were processed in parallel as negative controls. After washing in PBS, coverslips were mounted using the Prolong Antifade kit (Molecular Probes). Images were acquired with a SPOT digital camera attached to a Nikon Eclipse E800 or an Olympus DSU spinning disc confocal (Tokyo, Japan) microscope. Images were processed for printing using Photoshop 5.5 (Adobe Systems, San Jose, CA). Cells containing PMP22- and ubiquitin-positive aggregates (>1 µm in diameter), and the total number of cells (Hoechst positive), were counted in 8 to 10 random microscopic fields (0.1 mm²) using ImageJ software (NIH) and graphed as percentage of total cells (Fortun et al., 2007 (link)).