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ePaper created 2017-01-04, 09:21:41 | version 1.38.0
11 4 2016 laser research | and 65 °C. After a total of 15 min of thermal treat- ment,thecellswerecooleddownintheincubatorat a temperature of 37 °C for 1 hour. Some of the cells which had undergone thermal treatmentwereexaminedwiththeLife/DeadFluores cenc Assay (Molecular Probes, Eugene, OR, USA) in order to assess vitality via fluorescence microscopy and Axiovert 200 (ZEISS, Jena) after incubation. A mixture of 2 µM Calcein AM and 4 µM Ethidium- homodimer-D1 was added to the cells which were slowly cooling down at 37°C in the incubator either 1 hor24 hafterthermaltreatmentandincubatedfor 10'. Vital cells exhibited a green fluorescence caused by calcein, while lethal cells showed a red core fluorescence(Ethidium-homodimer-D1andcoupled DNA). 100 cells of each type were enumerated. In order to examine the synthesis of HSP proteins, the cells having undergone thermal treatment were processed as follows: – – Opening of the chamber and removal of the cover- slip containing the cells – – Suction of the nutritive medium, two rinses with PBS (isotonic: 67 mM phosphate buffer pH 7.2–7.4, 0.5 % NaCl) – – 12'fixationin2 %paraformaldehydein0.1Mcaco- dylatebufferpH7.2;Rinse:3xPBS,2xTBS(Trisbuff- ered saline, 50 mM Tris-HCl buffer, 1.25 % NaCl) – – PartingofthecoverslipwithPap-Penpen(oilpen), possibly correct with paraffin – – Incubate one half of the coverslip overnight at 4 °C with1:500dilutedantibodyAKHSP25,Rabbit(Bio- mol),dilutingsolution:fishgelatin1 %,Tritonx100 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:1:50withfishgelatine 1 % and Triton X 100 1% in TBS) – – Wash in TBS for 3x10' – – 15` Alkaline-Phosphatase verification with 3 mM Levamisol in Chedium (induces blue-brown co- louring 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' – – Washingwithcacodylatebufferfortwotimes,fol- lowed by two washings with Aqua dest – – Dehydration with increasing alcohol concentra- tion: 20 %, 30 %, 50 %, 70 %, 90 %, 2x in 100 % EtOH for 10' each – – FurtherprocessingofthesamplesattheCentrefor Electron Microscopy (Critical Point Drying and sputtering with gold; SCD 005, BAL TEC AG, Liech- tenstein) – – Microscope: Zeiss EM 902 A. Examinations with the transmission electron mi- croscope were conducted: – – Washingofthecellswithcacodylatebuffer(0.1 M) with 6.8 % Sucrose – – Fixation of 30' with 1 % glutaraldehyde – – Washing with cacodylate buffer – – Contrasting with 1 % Osmiumtetroxyde and 1 % potassium ferrocyanide for two hours – – Rinsing with cacodylate buffer for three times as well as with Aqua dest. – – Dehydration with increasing alcohol concentra- tion: 20 %, 30 %, 50 %, 70 %, 90 %, 2x in 100 % EtOH for 10' each – – Embedding in Epon (epoxy resin), polymerisation for four days at 60 °C – – Ultramicrotomy, ultra-thin sections (70 nm; Leica Ultracut S, Leica Mikrosysteme GmbH, Bensheim, Germany) Fig. 2 Fig. 2: HSP-detection caused by an antibody color reaction. 42016 1 % and Triton X 1001% in TBS)