Antifade

Mitotic metaphase and meiotic prophase I chromosome preparations were obtained as it follows: after treating bivalve specimens with colchicine (0.005%) for 12 h, gills and gonads were excised, immersed in 50% and 25% seawater for 1 h and fixed in a mixture of 75% absolute ethanol and 25% acetic acid for 1 h. Small pieces of fixed material were then treated with 60% acetic acid to obtain cell suspensions that were dropped onto slides heated to 50 °C^{41 ,42} .
Bivalve synaptonemal complexes were prepared from male specimens^{41 ,43 (link)}. Meiotic cell suspensions were dropped onto glass slides, treated with 0.1 M sucrose and 0.5% Triton X-100 for 2 to 4 min, fixed with paraformaldehyde (4%) overnight, rinsed with distilled water and air-dried.
Chromosome preparations were stained with a mixture of DAPI (0.14 µg/mL) and PI (0.07 µg /mL) for 8 min, washed in tap water, air-dried and mounted with antifade (Vectashield, Vector). Slide visualization and photography were performed using a Nikon Eclipse-800 microscope equipped with an epifluorescence system. Separated images for each fluorochrome were obtained with a DS-Qi1Mc CCD camera (Nikon) controlled by the NIS-Elements software (Nikon). Merging of the images was performed with Adobe Photoshop CS2 (Adobe Systems).

For early characterization of the CSC marker, CD133, immunofluorescent (IF) staining of spheres derived from U87 and U373 cell lines cultured in CSC medium was performed. For further analysis of U87 and U373 CSCs after drug treatment, IF staining was undertaken to investigate α-SMA, vimentin, and ß-catenin expression as previously described [43 (link)], with some modifications. Briefly, cells were fixed using 4% paraformaldehyde for 20 min at 4°C, and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were then incubated for 1h in dark conditions at 4°C with primary non-labeled mouse anti-human antibodies against CD133, α-SMA, vimentin, and ß-catenin (1:200 dilutions, BD Biosciences), followed by anti-mouse rabbit fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled secondary antibodies (1:500 dilutions, Invitrogen) for 1h at 37°C. The nucleus was counter-stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:10 000 dilution) and a drop of anti-fade (Vectashield; Vector Laboratory, Burlingame CA, USA) was added to avoid quenching of the fluorochrome before placing the cover slip. The slides were visualized using a Nikon Eclipse TE2000-U fluorescent microscope and photographs were taken using Qimaging- QICAM-fast 1394.

The beetles were killed with chloroform, and the dissected organs were put into Ringer’s solution to remove the trachea, Malpighian tubules and fat bodies. Spermiozeugmata were obtained by scratching out the seminal vesicles in Ringer’s solution. The genital organs were fixed in Carnoy’s solution, treated in an ascending ethanol series (70%, 80%, 96%) and isopropanol, and imbued with paraffin at 65°C. Using a Leica (SM 2010 R) microtome, the sample blocks were cut in 5- or 8-μm thin sections and then placed onto protein-glycerine treated slides. The deparaffinised samples were stained with haematoxylin and eosin (HE) after Romeis [33 ].
The aldehyde fuchsin-alcian blue (AF-AB) staining was applied after Spicer and Meyer [34 (link)]. For the alcian blue and periodic acid-Schiff (AB-PAS) staining, the samples were first treated with alcian blue, followed by periodic acid, Schiff’s reagent and haematoxylin. Then, the sections were embedded in Canada balsam. For DAPI staining the spermiozeugmata were placed onto polysine coated slides (Thermo Scientific) into a drop of Ringer’s solution. Then, 8 μl of 0.005% DAPI in Antifade (Vectashield) was added, and the sample was covered by an 18×18 mm coverslip.

Meiotic spread assays were performed to determine the identity of the freshly isolated pachytene spermatocytes from OA patients by STA-PUT velocity sedimentation. Briefly, cells were lysed by a hypotonic solution and spread evenly over slides layered with 1% PFA and 0.15% Triton X-100. Slides were dried for 24 hours at room temperature in a humid chamber. The cells were treated with 0.04% photoflo for 5 min and blocked with 4% normal serum. Double staining was performed in cells incubated with primary antibodies including SCP3 (Abcam) and CREST (Immunovision) overnight at 37°C in a humid chamber. Alexa 555 donkey anti-rabbit (Molecular Probes, Carlsbad, CA) and Alexa 488 goat anti-mouse (Molecular Probes) were used as the secondary antibodies and incubated for 90 min at 37°C. Cells were washed three times with TBS and treated with antifade (vector laboratories), and images were captured with a fluorescence microscope (Leica).

Japanese foxtail root tips were harvested from germinated seeds, pretreated with 2 mM 8-hydroxyquinoline for 4–6 h at 20°C to accumulate metaphase cells, and fixed in methanol–acetic acid (3:1) fixative. Root tips were macerated in 1 M HCl at 20°C for 1h and squashed in 45% acetic acid. All slides were stored at 70°C. After removing the coverslips, slides were dehydrated using 100% ethanol prior to the chromosome counting. Slides were stained in 4,6-diamidino-2-phenylindole (Roche Diagnostics, Switzerland) for 5 min at room temperature, and finally, anti-fade (Vector, USA) was applied under the coverslip. Slides were examined under an Olympus BX51 fluorescence microscope. Chromosome images were captured using an Evolution VF CCD camera (Media Cybernetics, USA) and merged using Image-Pro Express software.

Slides with sorted chromosomes were blocked using a blocking solution (5% BSA, 0.03% Triton X-100, 1× PBS) for 1 h at RT and incubated with primary antibodies against Topo II diluted (1:100) in antibody solution (1% BSA, 0.01% Triton X-100, 1 × PBS) overnight at 4 °C. Next, after washing in 1 × PBS, slides were incubated with secondary donkey anti-rabbit Alexa488 antibodies (1:200, #711-545-152 Jackson ImmunoResearch), diluted in antibody solution, for 1 h at 37 °C. Subsequently, the slides were washed in 1 × PBS at RT and immediately dehydrated in an ethanol gradient (70%, 85%, and 100%), each step 1 min. Next, slides were air-dried, counterstained with 1 µg/mL DAPI in antifade (Vectashield, Vector Laboratories, Burlingame, CA, USA), and subjected to microscopy.

Expression of fucosyl glycoconjugates on the mucosal surface was measured on frozen tissue sections using FITC-conjugated Ulex europaeus agglutinin-1 (UEA-1; Vector Laboratories, Burlingame, CA, United States). The middle 1 cm of the colon was fixed for 4 h at 4°C in 4% paraformaldehyde, washed in ice-cold PBS containing 30% sucrose overnight at 4°C, and embedded in optimal cutting temperature compound. Frozen sections (6–7 μm thick) were blocked with PBS containing 2% BSA and then stained with labeled lectin for 1 h (10 μg/ml). Sections were then washed three times in ice-cold PBS, mounted using Anti-Fade (Vector Labs), and analyzed by confocal microscopy (9 (link)).