Araldite ddsa mixture

The ultrastructural changes in the cell components of virus infected poppy plants were studied using transmission electron microscope (TEM). Briefly, leaf samples of healthy and diseased samples were washed with 1X PBS (pH 7.2) prior to fixing in 2.5% glutaraldehyde prepared in sodium cacodylate buffer (pH7.2) for 2 h at 4°C. Samples were washed three times with 0.1 M sodium cacodylate buffer and post fixed in 1% osmium tetroxide for 2 h. Samples were washed with sodium cacodylate, dehydrated in acetone series (15–100%) and embedded in Araldite-DDSA mixture (Ladd Research Industries, United States). After baking at 60°C, blocks were cut (60–80 nm thick) by an ultramicrotome, (Leica EM UC7), and sections of leaf of healthy and infected plant samples were stained with uranyl acetate and lead citrate, and analyzed under G2 spirit twin TEM equipped with a Gatan digital CCD camera (FEI Tecnai, The Netherlands) operating at 60 or 80 KV. The region selected for microscopy was near the phloem tissue where the possibility of virus accumulation was highest.

The ovules were cultured at −3 DPA and treated with inhibitors. At 1 DPA the control and treated ovules were washed with 1XPBS (pH 7.2). The ovules were then fixed in 2.5% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer (pH 7.2) (Ladd Research) for 4 h at 4 °C followed by three times washing with 0.1 M sodium cacodylate buffer. Further, the ovules were treated with 1% osmium tetraoxide for 4 h and thoroughly washed with sodium cacodylate. After this, the ovules were dehydrated in acetone series (15–100%) and then embedded in araldite-DDSA mixture (Ladd Research Industries, USA) followed by baking at 60 °C. Ultra-microtome (Leica EM UC7) was used to cut 60-80 nm thick sections from the blocks. Further, these sections were stained with uranyl acetate and lead citrate and analyzed under FEI Tecnai G2spirit twin transmission electron microscope equipped with Gatan digital CCD camera (Netherland) at 80 kV.

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.