Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labelled ribonucleotide probes to embedded tissue.
In an attempt to identify key changes involved in normal spermatogenesis we have developed methods to enable the study of gene expression by the various subpopulations of testicular cells by use of in-situ hybridisation histochemistry. The use of digoxigenin-labelled ribonucleotide and oligonucleotide probes on testicular tissue perfusion-fixed with Bouin's fixative and embedded in paraffin, polystyrene or methacrylate, has been used to accurately localise three transcripts to three different cell types (Sertoli cells, pachytene spermatocytes, and step 7-12 spermatids) within the seminiferous tubule. The ability to produce semi-thin sections of polystyrene- or methacrylate-embedded tissue and successfully to apply digoxigenin-labelled ribonucleotide or oligonucleotide probes resulted in far greater resolution and unequivocal localisation of mRNA in testicular cells than was previously possible by use of thicker paraffin or frozen sections hybridised with 35S-labelled riboprobes. A comparison of the different embedding media versus digoxigenin-labelled oligonucleotide or ribonucleotide probes is made and we demonstrate the relative sensitivities and merits of each combination.
Millar
M R
MR
M.R.C. Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK.
Sharpe
R M
RM
Maguire
S M
SM
Saunders
P T
PT
D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D003546:Cysteine Endopeptidases; D004247:DNA / Q000235:genetics; D004076:Digoxigenin; D003576:Electron Transport Complex IV / Q000235:genetics; D010450:Endopeptidases; D005069:Evaluation Studies as Topic; D008297:Male; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013737:Testis / Q000166:cytology / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger; C053886:spermatid transition proteins; D004247:DNA; D003576:Electron Transport Complex IV; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat; D004076:Digoxigenin