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.

Localization of 15K mRNA in the Male Gonad by In Situ Hybridization

M. Klass, M. Herndon

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.