17 Introduction to the Detection of Transcription Regulatory Proteins
239
a
c
e
9
h
A
p
A
p
Fig.2. In situ localization of POMe mRNA in the paraffin sections of rat pituitary gland. As described in the text, the paraffin sections of rat pituitary gland
were hybridized in situ with DNP-Iabeled POMC cDNA (a, c, e, g) and DNPlabeled pBR 322 DNA (b, d, f, h) and the signal was detected enzyme-immunohistochemically (Koji et al. 1988). In this experiment, the effects of increasing
concentrations of proteinase K (37°C, 15 mins) on the signal detection was investigated, since the optimization of proteinase digestion conditions is the most
important factor in achieving the best performance of nonradioactive in situ
hybridization (c.f., Chapter 15). (a, b); 0 Ilg/ml, (c, d); 1 Ilglml, (e, f); 10 Ilg/
ml, (g, h); 100 Ilg/ml. A; anterior pituitary, I; intermediate pituitary, P; posterior
pituitary. At 100 Ilg/ml of proteinase K, the specific signal for POMC mRNA was
observed in the intermediate pituitary cells and some cells of anterior pituitary.
However, a clear staining with the similar distribution pattern to that ofPOMC
mRNA was obtained with pBR 322 DNA at lower concentrations of proteinase K.