Hybridization histochemistry

Abstract

In this review we have used our own recent work as a flagship to illustrate the recent renaissance of interest in hybridization histochemistry. A trickle of papers followed the initial key excursion into the in situ labeling of tissue sections (48-50). Our own entry into this field started in 1978 and since then a confluence of important questions and technical advances has served to make hybridization histochemistry much more attractive as a research tool. Hybridization histochemistry is able to solve some problems for which there is no other suitable technique at this time. Hybridization histochemistry provides the location of anatomical sites of gene expression, and viral replication, with uniquely high specificity. We have taken 32P-labeled probes to what appears to be their limit of resolution, which is single cells in thin sections. While 32P has clear disadvantages, exposure time is relatively short and the use of fast-X-ray film to preview the results and estimate exposure time for emulsion has been turned to advantage. Our introduction (27) of the use of whole-mouse sections in hybridization histochemistry has great potential in hormonal, enzymatic, and growth factor gene expression and will no doubt prove of great use in developmental studies and examination of viral infection. The use of synthetic DNA (synthetic oligonucleotides) unshackles the technique from the need for an associated molecular biology laboratory and at once widens the horizon of application of the technique. Although hybridization histochemistry is a valuable research tool which will soon find a niche in many fields, in a short time it should become a key diagnostic aid. It may well become the method of preference for detection of the expression of oncogenes and other cancer-related genes and for viruses which for other reasons are difficult to detect.

Substance P – a potent risk factor in childhood lymphoblastic leukaemia

“…16 For detection of mRNA encoding the amino-acid sequence of human SP, a 5 0 -biotinylated probe of the following nucleotide sequence: 5 0 GAG TTT GGA TTC TGA TGA CCT CCC AAG CCG GCA 3 0 , was used. 17 The probe was synthesised by the DNA-Gdań sk company and was complementary to the nucleotide sequence of human SP (GenBank; www.ncbi.nlm.nih.gov). The smears were incubated with the probe (concentration: 200 ng/1 ml) for 18 h at +371C in a hybridisation chamber.…”

Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus

“…Oligonucleotide probes can be prepared in a few days, are easier to label than cloned probes, and give lower background (Coghlan et al 1985). These probes can be ordered already biot~nylated, or in a gel purified form ready for labeling.…”

“…Biotinylated oligonucleotide probes are well suited for in situ hybridization using tissue sections from infected animals (Coghlan et al 1985, Cubie & Norval 1988. Thls technique, in con~bination with the PCR, has significant application for research into the carrier state of IHNV, as it has the potential to detect nucleic acids of viruses in a latent state (Maitland e t al.…”

In situ localization of c-myc mRNA in HL-60 cells using non-radioactive synthetic oligodeoxynucleotide probes.

“…Conventionally using cloned double-stranded cDNA as a probe , a lot of studies to establish a tissue processing procedure best suited for in situ hybridization have been done (6,22,39). As already stated, however, the advantage of the use of doublestranded cDNA is limited by the presence of sense strand, whose sequence is identical to that of the corresponding mRNA.…”

“…To accomplish this task, the localization of mRNA by means of in situ hybridization was found to be the most effective method (6,22,39).…”