Panoramic midi 2 slide scanner

Paraffin sections were stained with a pH3 antibody (rabbit polyclonal, Cell Signaling Technology, 9701, 1:400), anti SOX9 (Sigma-Aldrich, HPA001758, 1:200), anti Col2A1 (Developmental Studies Hybridoma Bank, CIIC1-b, 1:250) and anti-Myosin II (Developmental Studies Hybridoma Bank, CM1123, 5 µg/ml). Standard antigen retrieval with DIVA Decloaker (BioCare Medical) was carried out. Species-specific secondary antibodies tagged with Alexa Fluor 488 or Cy5 were used (goat anti-mouse 488, A11029; goat anti-rabbit 488, A11034; goat anti-mouse Cy5, A10524, Life Technologies; all at 1:250).
Apoptosis was assessed by TUNEL analysis using the ApopTag Apoptosis Kit (Millipore, S7111) and was detected using fluorescein-tagged anti-digoxygenin antibody as described by Hosseini-Farahabadi et al. (2013) (link). A qualitative assessment of presence of TUNEL-positive cells was carried out.
Nuclei were counterstained in Hoechst 33258 (Sigma-Aldrich, 10 µg/ml in PBS) for 30 min. Slides were mounted with Prolong Gold without DAPI (Invitrogen, P36934). Fluorescence imaging was conducted using a 20× objective on a Panoramic MIDI II slide scanner with 488, Cy5 and DAPI filter sets (3D Histech). High-magnification images were captured with CaseViewer software v.2.4.0.119028 (3D Histech).

All testicular slides were scanned using the Panoramic MIDI II slide scanner (3DHISTECH, Hungary). The histomorphometric analyses were performed using the CaseViewer software (3DHISTECH, Hungary) and the Image J v.1.45s software (Image Processing and Analysis, in Java, USA).

Freshly dissected embryos were fixed in X-gal buffer containing 0.2% glutaraldehyde and 1% formaldehyde on ice for 15 min and subjected to modified protocol from Tremblay 2000 [16 (link)].In brief, the fixed embryos were washed with X-gal buffer (PBS, 5 mM EGTA, 2 mM MgCl:6H2O, 0.2% NP-40, 0.2 mM deoxycholate) for 10 minutes three times, and stained with X-gal stain (X-gal buffer, 5mM potassium ferricyanide and 5mM potassium ferrocyanide and 0.5 mg/ml X-gal) overnight at 37°C. Subsequently, embryos were dehydrated in ethanol, cleared in xylene, embedded with paraffin, and sectioned at 7μm thick. The sectioned embryos were deparaffinated and rehydrated for subsequent processing. Eosin staining was performed by immersing rehydrated sectioned embryos in eosin Y solution for 15–20 seconds, followed by 95% ethanol for 2 minutes and then 100% ethanol for 2 minutes, and lastly cleared in xylene. Slides were then sealed with Cytoseal 60. X-gal stained sections were imaged with a Panoramic MIDI II slide scanner (3dHistech).