5-1植物组织印染法.doc

5-1 植物組織印染法 在硝酸纖維紙上做組織印染 將新鮮的組織切口壓印在硝酸纖維紙上20-30秒，隨即將印染過的紙放入80℃抽真空之烘箱中約兩小時後即可染色或在顯微鏡下觀察。在印染的時候，對每個材料所施的壓力求取一致是很重要的。 2007/3/8 生物顯微實驗技術 上課前準備清單 80℃抽真空之烘箱 蒸餾水 光學顯微鏡*1 解剖顯微鏡*1 5-2 In situ Hybridization 1. Tissue Fixation in FAA A. FAA (formalin-aceticacid-alcohol) for 100 ml 90%EtOH 50 ml glacial acetic acid 5 ml formaldehyde (37%) 10 ml ddH2O 35 ml B. Dehydration using Histochemical processor and embedded in 62℃ melted paprffin 70% (in DEPC ddH2O) 30 min 80% (in DEPC ddH2O) 30 min 95% 30 min 100% 30 min x 3 Hemo-D 2 hr paraffin infiltration for several days 7 days embedded and solidification C. Cut the sections by new knife and put the sections directly onto the slide which was placed on heat block with one drop of DEPC-treated ddH2O. Let the section rehydeate and dry on the slide. D. De-Paraffin: Use slides with 1 or 2 sections close together (this reduces the amount of solutions needed). Deparaffinization: 2x, 5m - Hemo De 2x, 5m - 100% EtOH 1x, 5m - 50% EtOH (in DEPC ddH2O)  air dry completely, circle sections with rubber cement to keep solutions on sections and for sealing coverslip. 2. Probe preparation: A. plasmid construction: insert the desired cDNA into pBluescript vector (SK-) (because the promoters used are T3 and T7 promoters in pBluescript vector) B. template DNA preparation: a. linearization of template DNA by restriction enzyme digestion (in DEPC H2O) b. check the linearity of the DNA by agarose gel electrophoresis c. Phenol/chloroform - add more TE buffer to final volume to 400 ml - add 200 ml of phenol and 200 ml chloroform, shake well - spin max speed x 5 min - take the aqueous phase (top fraction) into new microfuge tube - add 400 ml chloroform and shake well and spin max spped x 5 min - the the top fraction into new microfuge tube - ppt the DNA: add 1/10 final volume of NaOAc - for isoproponal: add 200 ml isopr