TY - JOUR
T1 - Efficiency of in situ hybridization as a function of probe size and fixation technique
AU - Moench, Thomas R.
AU - Gendelman1, Howard E.
AU - Clements, Janice E.
AU - Narayan, Opendra
AU - Griffin, Diane E.
N1 - Funding Information:
This work was supported by Public Health Service Grants I-POl-NS-15721, NS-16145, and NS-07000. We thank Dr. Shmuel Rozenblatt, Weizmann Institute, for providing measles virus clone N. We thank Linda Kelly for secretarial assistance.
PY - 1985/6
Y1 - 1985/6
N2 - In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.
AB - In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.
KW - in situ hybridization fixation periodate-lysine-paraformaldehyde-glutaraldehyde
UR - http://www.scopus.com/inward/record.url?scp=0021836698&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021836698&partnerID=8YFLogxK
U2 - 10.1016/0166-0934(85)90035-7
DO - 10.1016/0166-0934(85)90035-7
M3 - Article
C2 - 2991313
AN - SCOPUS:0021836698
SN - 0166-0934
VL - 11
SP - 119
EP - 130
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 2
ER -