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ePaper created 2017-06-21, 10:00:36 | version 1.38.0
temperature-related damage of the DPSC study | Fig. 1 Examinations with the transmission electron mi- Fig. 1: Vitality test of thermally treated croscope were conducted: · Washing of the cells with cacodylate buffer (0.1 M) DPSC. with 6.8 % Sucrose · Fixation of 30' with 1 % glutaraldehyde · Washing with cacodylate buffer Fig. 2: HSP-detection caused by an antibody colour reaction. Fig. 2 roots 2 2017 31 · Suction of the nutritive medium, two rinses with PBS (isotonic: 67 mM phosphate buffer pH 7.2–7.4, 0.5 % NaCl) · 12' fixation in 2 % paraformaldehyde in 0.1 M cacodylate buffer pH 7.2; Rinse: 3 x PBS, 2 x TBS (Tris buffered saline, 50 mM Tris-HCl buffer, 1.25 % NaCl) · Parting of the coverslip with Pap-Pen pen (oil pen), · possibly correct with paraffin Incubate one half of the coverslip overnight at 4 °C with 1:500 diluted antibody AK HSP25, Rabbit (Biomol), diluting solution: fish gelatin 1 %, Triton x 100 1 % in TBS) · Cover the other half of the coverslip exclusively in diluting solution (without AK) · Wash in TBS for 3 x 10' · Conjugate with the second antibody AK Anti-Rab- bit-Alkaline Phosphatase for two hours at room temperature (Ziege, dilution: 1:50 with fish gela- tine 1 % and Triton X 100 1 % in TBS) · Wash in TBS for 3 x 10' · 15' Alkaline-Phosphatase verification with 3 mM Levamisol in Chedium (induces blue-brown colour- ing according to Seidel). In order to perform examinations with scanning electron microscope, the cells were processed as fol- lows: · Washing of the cells in cacodylate buffer (0.1 M) · Fixation with 2.5 % Glutaraldehyde in cacodylate buffer for 20' · Washing with cacodylate buffer for two times, fol- lowed by two washings with Aqua dest. · Dehydration with increasing alcohol concentration: 20 %, 30 %, 50 %, 70 %, 90 %, 2 x in 100 % EtOH for 10' each · Further processing of the samples at the Centre for Electron Microscopy (Critical Point Drying and sputtering with gold; SCD 005, BAL-TEC AG) · Microscope: Zeiss EM 902A.