Cellsens image analysis software

Three mice were perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Then, the skull was post-fixed for 10 h, decalcified in PBS containing 250 mM EDTA for 7 days, cryo-protected by overnight incubation in PBS containing 30% w/v sucrose, mounted in OCT Embedding Compound (Sakura Finetek, Tokyo, Japan) and frontally sectioned at 25-μm thickness using a cryostat (CM3050S, Leica Microsystems, Wetzlar, Germany). Sections were then incubated at room temperature overnight in an appropriate blocking solution containing an anti-CAPN5 primary antibody (ab28280, Abcam, CA, USA), washed with PBS containing 0.3% Triton X-100 (PBS-X), incubated with DAPI and the appropriate secondary antibody (Alexa-Fluor 594 conjugated, 1:200; Molecular Probes, USA; in blocking solution) for 1.5–2 h, washed in PBS-X, mounted onto MAS-coated glass slides (Matsunami Glass, Osaka, JAPAN), coverslipped using Vectashield antifade reagent (Vector Labs, CA, USA) and tightly sealed. The fluorescence signals were detected using a fluorescent microscope (BX50; Olympus, Tokyo Japan) and were analyzed with cellSens image analysis software (Olympus, Tokyo, Japan). The images for CAPN5-IR were captured by exposing the samples for 40 ms at low magnification (× 10, objective) and for 80 ms at high magnification (× 60) and were analyzed offline with NIH ImageJ (MD, USA).

Samples preserved for light microscopy were rinsed for 2h in distilled water and dehydrated through an ascending series of ethanol (70%, 96%, 100%) and xylene. Samples were then embedded in paraffin at 60°C overnight and cut with a Microtome Micron HM325 to 5 μm sections. Staining was performed using both Methylene blue and Hematoxilin-Eosin standard protocols. Pictures of reproductive elements were obtained with a microscope Olympus BX43 and an SC50 5MP colour CMOS camera. The counting and measurements (maximum diameter) of reproductive elements in each section were performed with the CellSens image analysis software of Olympus.
Samples preserved for TEM were subjected to a protocol of rinsing, fixation, dehydration and embedding following [9 ]. The sections of Spurr resin blocks were performed at 64 nm using an ULTRACUT ultramicrotome, stained with lead citrate and uranyl acetate. They were observed with a JEOL 1010 electron microscope with a Gatan module for image digitalization at the Microscopy Unit at the Scientific and Technological Centres, Universitat de Barcelona (CCiT-UB).