Ccd digital camera

The testes dissected from the control and knockdown males from both ERR lines (ERR-miRNA & ERR-TRiP) and those dissected from pupae of Sox100B-TRiP lines were incubated, independently, in fixing solution (4% Paraformaldehyde, 2% Glutaraldehyde, 0.05% Sucrose, and 0.1% Sodium Cacodylate) for 90 min. The tissues were washed thrice in 0.1 M Sodium Cacodylate for 20 min each, prior to overnight incubation in 1% Osmium tetraoxide (OsO₄). The tissues were then washed thrice in 0.1% Sodium cacodylate for 20 min each and were treated with 1% OsO₄ for 2 h. These tissues were sequentially dehydrated in 15%, 30%, 60% and 100% acetone, respectively, for 20 min. Subsequently, tissues were embedded in a low viscosity epoxy resin araldite medium to prepare blocks. These blocks were used to cut semi and ultrathin sections using a ultra microtome (EM-UC7 ultramicrotome, Leica, Germany). Sections were stained in Toluidine blue staining solution. The sections were examined in a Tecnai G2 spirit TWIN transmission electron microscope (FEI, USA), equipped with digital CCD camera (Gatan, Netherland, Europe) and the structural details of testicular sections were recorded (1 μm, 15000X, and 0.5 μm, 30000X). The electron microscopic analysis was performed thrice per batch (control/ERR-miRNA/ERR-TRiP/Sox100B-TRiP) with each batch having a minimum of five replicates (pairs of testes).

Fungus was grown in minimal media with sodium arsenate (As V) at 0.1 and 0.5 mM concentration while control was grown without arsenic. After 3 days of growth, mycelia was filtered and washed 10 times with autoclaved MQ water. Further, fungal mycelium of control and treated were washed with 1XPBS (pH 7.2) and fixed in 2.5% glutaraldehyde prepared in phosphate buffer (pH 7.4) for 2 h at 4°C. Cells were washed three times with 0.1 mM phosphate buffer and post fixed in 1% Osmium tetroxide for 4 h. Fixed cells were washed with phosphate buffer, dehydrated in acetone series (15–100%), and embedded in Araldite-DDSA mixture (Ladd Research Industries, USA, Burlington). After baking at 60°C, block was cut (60–80 nm thick) by an ultramicrotome (Leica EM UC7) and sections were stained with Uranyl acetate and Lead citrate. Analysis of sections was done under FEI Tecnai G2 spirit twin transmission electron microscope equipped with Gatan digital CCD camera (Netherlands) at 80 KV. The confirmation of insoluble precipitates as arsenic was done by SEM (Scanning electron microscope) equipped with EDAX.

Isolated EVs from plasma and serum were re-suspended in PBS and placed on a formvar-coated copper grid and then allowed to settle for 45 min. Sequential PBS washing of the grid was done by positioning droplets of PBS on the top and applying absorbing paper in between. The samples were fixed by positioning the grid on the top of 2% paraformaldehyde placed on the parafilm for 15 min. After fixation and three washing steps, samples were contrasted by adding drops of 2% uranyl acetate for 15 min. A drop of 0.13% methyl cellulose and 0.4% uranyl acetate was placed on the parafilm grid was incubated at the top for 10 min. The grid was visualized by a Philips CM10 transmission electron microscope and images were recorded using a Gatan CCD digital camera.