Worm Breeder's Gazette 7(1): 54
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have done an in situ hybridization analysis on dissected male tissues by using a 720bp Xba genomic fragment that is complementary to 15K mRNA as a probe. Hybridization was confined to a specific region of the male gonad in the mid-proximal arm beginning approximately one fourth the distance past the loop region. This is the same region that the sperm-specific protein 15K is first detected by immunocytochemistry.Hybridization was sensitive to RNase pre-treatment and was not observed in spermatids or in any other tissues of the male. DNase pre-treatment had no effect on hybridization. The conditions for in situ hybridization were as follows: 1) Tissues were dissected from adult males in M9 salt solution on gelatinized glass slides and fixed by the addition of 45 acetic acid 2) Tissues were flattened (not squashed) by the pressure of a cleaned coverslip; coverslips were subsequently removed by dipping in liquid nitrogen 3) Post fixation was in 3:1 ethanol: acetic acid 4) Slides were dehydrated to 100% ethanol at -20 C, air dried and baked at 80 C for 2 hrs in a vacuum oven. 5) Hybridization conditions were the same as those used by Thomas ( PNAS 77, 5201-5205, 1980) for the Northern blot. Pre-treatment was in 50% formamide, 5 X SSC, 50mM NaP04 pH 6.5, 250mg/ml sheared E. coli DNA, 0.02% w/v BSA, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrolidone at 42 C for 1 hr. Hybridization solution consisted of pre-hybridization solution plus 10% dextran sulfate, 2 mg/ml of Poly A and the labeled probe ([3H]p lambda8 1 X 10+E6 cpm/slide; 0.5-1.0 X 10+E7 cpm/ug). Hybridization was carried out overnight at 42 C by adding 35-40ml of hybridization solution to each slide, covering with a cleaned coverslip and sealing with Sanford's Rubber contact cement. 6) Post-Hybridization washes were carried out by first gently removing the coverslip and washing in 2 changes of prehybridization buffer without DNA for 10 minutes each at 42 C followed by four washes in 2 X SSC for five minutes each at room temperature. The slides were then washed twice in 0.1 X SSC at 50 C for 15 minutes each followed by washing in distilled water, washing in 5% TCA at 0 C and finally in distilled water and air dried. Squashing the tissue caused severe tissue destruction and a great increase in background probably due to the leakage of the cellular contents (mRNA?) by the gonad. Pre-treatment with proteinase-K caused an increase in the loss of tissue even after baking at 80 C. Washing in 0.1 X SSC at 50 C after hybridization was necessary to reduce background. Sealing the coverslip with rubber cement was suggested by Dr. W. Jeffery and was necessary to prevent evaporation. Removal of the sealed coverslip after hybridization often caused tissue loss due to the rubbing of the coverslip on the tissue. This was prevented by suspending the coverslip over a single layer of Scotch brand tape surrounding the tissue area.