Phosphotungstic Acid

The standard chromosome spreading protocol has been described previously for our laboratory (Lenzi et al., 2005 (link)). The nuclear contents of whole-mount spermatocytes (or oocytes) were displayed by drying down a cell suspension, in hypotonic buffer, from either testis, or ovary, in 1% paraformaldehyde containing 0.15% Triton X-100 (Peters et al., 1997 (link)). Whole testes or ovaries were incubated on ice for 60 min in hypotonic extraction buffer (HEB; 30 mM Tris, pH 8.2, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). Either a one-inch length of tubule, or a whole ovary, were placed in a 20-μl drop of 100 mM sucrose, pH 8.2, the tissue was macerated, and a second 20-μl drop of sucrose solution was added and the cell suspension was pipetted up and down several times. Remnant pieces of tubule were removed. Cleaned slides were dipped in the paraformaldehyde and Triton X-100 solution, and most liquid was drained off, such that only enough liquid remained to coat the slide. 20 μl of the cell suspension was added in one corner and the cells were slowly dispersed, first in a horizontal direction and then vertical. The remaining 20 μl of cell suspension was used to make a second slide and both were placed in a humid chamber to dry slowly at RT for 2 h. The slides were washed three times for 1 min in 0.4% Kodak Photo-Flo 200 and air dried for at least 15 min. For EM preparations, to make the SCs accessible to immunogold grains, the slides were DNaseI treated (1 μl/ml of DMEM) before being air dried (Moens et al., 2002 (link)). The slides were washed and blocked (three times for 10 min each) in PBS and incubated in primary antibodies overnight at RT in a humid chamber. Primary antibodies were used at varying concentrations, and generally a 10-fold higher concentration was used for EM than immunofluorescence. After washes, slides were incubated in secondary antibodies, conjugated to either fluorochrome or colloidal gold (Jackson ImmunoResearch Laboratories), for 2 h at 37°C. After washes the slides were mounted with ProLong Antifade (Invitrogen) for fluorescence microscopy. Images were captured on a Olympus IX81 microscope attached to a 12-bit Cooke Sensicam CCD instrument and sent to IP Lab software.
For EM, slides were incubated in 4% alcoholic phosphotungstic acid for 15 min, followed by three 1-min washes in 95% ethanol, to enhance visualization of MNs. Slides were air dried and then dipped in 0.25% formvar (Electron Microscopy Sciences) and air dried under glass. The plastic was scored, treated with 25% hydrofluoric acid, and floated off in water with attached cells. Plastic was transferred to EM grids and used for transmission EM (JEOL 1200EX).

The MDA-MB-231, SUM149, SUM159 and EMT6 cell lines were treated with different concentrations of GO (3.550, 3.550, 1.437 and 2.22 mmol/l, respectively) for 24 h. For TEM, a total of 1x10⁷ cells were pelleted by centrifugation at 2,683 x g for 5 min at 26˚C, then washed three times with PBS. The cells were then fixed in 2.5% glutaraldehyde at 4˚C for 24 h. Next, the cells were washed with PBS three times and post-fixed in 1% osmium tetroxide for 60 min at 4˚C, encapsulated in 1% agar and stained with uranyl acetate and phosphotungstic acid for 60 min at 4˚C. The cells were then dehydrated in a graded ethanol series and subsequently incubated in propylene oxide for 35 min at 26˚C. The TEM images were captured using a Hitachi TEM system (Hitachi High-Technologies Corporation).
For 3D micro-morphology, the volume and height of the SUM149, MDA-MB-231, SUM159 and EMT6 cell lines were measured using a VK-V150 laser microscopy system (Keyence Corporation). Phase-contrast observations of the cells were performed using an Olympus IX71 microscope (Olympus Corporation). The results were obtained from three independent experiments.