Polylysine coated slides

Testes and epididymides were embedded in paraffin, sectioned (5 μm), mounted on polylysine-coated slides (VWR, Leuven, Belgium), and stained with haematoxylin and eosin according to standard procedures for morphological analysis. Immunohistochemistry was performed only on testis sections. For this, we used the ABC Kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions. For single and double immunofluorescence, preparations were incubated overnight with primary antibodies, washed, incubated with the appropriate conjugated secondary antibodies for 1 h at room temperature and treated with 4′,6-diamino-2-phenylindol (DAPI). Digital photomicrographs were taken in a Nikon Eclipse Ti microscope (Nikon Corporation, Tokio, Japan). Three testicular sections from each of the groups of males of both species were mounted on the same slide and processed together. The primary antibody was omitted in negative controls. Supplementary Table S3 summarises the antibodies and working concentrations used in this study.

J-Lat 1C10 cells were washed with PBS and allowed to adhere to Polylysine coated slides (VWR, Radnor, PA, USA) marked with a hydrophobic barrier using A-PAP pen (Histolab, Gothenburg, Sweden). PLA was performed according to the manufacturer’s protocol (Sigma) with a few modifications: PLA plus and minus probes were diluted 1:20, amplification buffer (5×) was used at 10×. All washes were performed in PBS. Antibodies (1:1,000) were used against Tat (Abcam, Cambridge, United Kingdom), FLAG M2 (Sigma). Before DAPI staining and mounting with Duolink in Situ mounting media with DAPI (Sigma), FITC-conjugated anti-GFP (Abcam) was applied (1:500) for 1 h at ambient temperature protected from light. Slides were sealed with nail polish and stored at 4°C overnight before imaging. Slides were imaged using a Pannoramic Midi II slide scanner (3DHistech, Budapest, Hungary) and images were exported using the CaseViewer application. Images were analyzed with ImageJ (version 2.0.0-rc-69/1.52) and macros developed in house. Each RGB image was separated into separate channels. Based on the blue (DAPI) channel, nuclei were identified. In the red channel, spots (“maxima”) were detected with prominence thresholds 6–48.

The hypoxic status was determined using the Hypoxyprobe^TM-1 Plus Kit supplied by Hypoxyprobe Inc (Burlington, MA, USA) following the supplier's instruction. For immunocytochemistry assay, the cells were cultured in 8-well chamber slides at normoxic (20% oxygen) or hypoxic [1% oxygen in Hypoxic Chamber (StemCell, Durham, NC, USA)] condition for 24 hours and labeled with Hypoxyprobe for 2 hours. The hypoxic cells were detected by confocal microscope after stained with FITC-conjugated anti-hypoxyprobe MAb. The CSCs and cells in suspending condition were cultured for 6 days. The MSCs and suspension-cultured cells were labelled with Hypoxyprobe for 24 hours and cytospined at 800 rpm for 3 min to spread the spheres onto Polylysine-coated slides (VWR, Lutterworth, UK). For flow cytometric analysis, the cells were cultured in 25cm flasks at the above conditions. After immunocytochemistry staining the cells were subjected to flow cytometric analysis. The hypoxic population was detected using a FACSCalibur flow cytometer with 488-nm blue laser and standard FITC 530/30 nm bandpass filter. To determine the effect of hypoxia on drug sensitivity, the cells were cultured in 1% oxygen condition at a cell density of 1×10³ cells/well in 96-well plate for 4 days and exposed to anticancer drugs for another 72 h before MTT assay. The parallel MTT assay was performed in normoxic condition.