Summary
Non-radioactive in situ hybridization using biotinylated oligodeoxynucleotides and a detection protocol involving monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase system was employed for quantitation by image analysis. Calibrations of the image analysis system with neutral density filters revealed that the grey levels recorded were strongly linearly correlated to the absorbance (r 2=0.97) in the range studied in tissue specimens (0–0.8 optical density or absorbance units). Several methodological parameters, including light source stability, section thickness, probe concentration and development time were initially optimized. Model systems revealed that the grey level measured varied linearly with the logarithm of the target concentration. Moreover, histophysiological studies on adrenalectomized and sham-operated rats documented that previous biochemical data on an 8- to 10-fold increase in anterior lobe proopiomelanocortin (POMC) mRNA levels 8 days after adrenalectomy are accounted for both by an increased ACTH cell concentration and content of POMC mRNA, as well as by increases in ACTH cell sizes and cell numbers. Also in agreement with biochemical data, image analysis did not reveal significant differences between OD's of melanotrophs in adrenalectomized and sham-operated animals. To our knowledge, these data are the first to document that non-radioactive in situ hybridization can be employed for relative quantitation. A particular advantage of this approach is the good morphological definition which permits parallel analyses of densitometric values, cell sizes and cell areas/cell numbers.
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Autelitano DJ, Blum M, Roberts JL (1989) Changes in rat pituitary nuclear and cytoplasmic pro-opiomelanocortin RNAs associated with adrenalectomy and glucocorticoid replacement. Mol Cell Endocrinol 66:171–180
Burbach JPH, Voorhuis TAM, Tol HHM van, Ivell R (1987) In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups. Biochem Biophys Res Commun 145:10–14
Butcher RG (1972) Precise cytochemical measurement of neotetrazolium formazan by scanning and integrating microdensitometry. Histochemie 32:171–190
Butcher RG (1978) The measurement in tissue sections of the two formazans derived from Nitroblue Tetrazolium in dehydrogenase reactions. Histochem J 10:739–744
Childs GV, Yamauchi K, Unabia G (1989) Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes. Am J Anat 185:223–235
Civelli O, Birnberg N, Herberg E (1982) Detection and quantitation of pro-opiomelanocortin mRNA in pituitary and brain tissues from different species. J Biol Chem 257:6783–6787
Fremeau RT Jr, Lundblad JR, Pritchett DB, Wilcox JN, Roberts JL (1986) Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei. Science 234:1265–1269
Hatfield JM, Daikh DI, Adelman JP, Douglass J, Bond CT, Allen RG (1989) In situ hybridization detection of marked differences in pre-proopiomelanocortin messenger ribonucleic acid content of individual corticotropes and melanotropes. Endocrinology 124:1359–1364
Isseroff A, Lancet D (1985) An inexpensive microcomputer-based image-analysis system: novel applications to quantitative autoradiography. J Neurosci Methods 12:265–275
Jarvis LR (1988) Microcomputer video image analysis. J Microsc 150, pt. 2:83–97
Larsson L-I (1988) Immunocytochemistry: theory and practice. CRC Press, Boca Raton, Fl., USA
Larsson L-I (1989) In situ hybridization of messenger RNA sequences. Histochem J 21:435–440
Larsson L-I, Christensen T, Dalbøge H (1988) Detection of proopiomelanocortin mRNA by in situ hybridization using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry. Histochemistry 89:109–116
Larsson L-I, Hougaard DM (1990) Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions. Histochemistry 93:347–354
Lawrence GM, Beesley ACH, Matthews JB (1989) The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity. Histochem J 21:557–564
Leary JJ, Brigati DJ, Ward D (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio-blots. Proc Natl Acad Sci USA 80:4045–4049
Lewis ME, Krause II RG, Robert-Lewis JM (1988) Recent developments in the use of synthetic oligonucleotides for in situ hybridization histochemistry. Synapse 2:308–316
Mize RR, Holdefer RN, Nabors LB (1988) Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis. J Neurosci Methods 26:1–24
Nabors LB, Songu-Mize E, Mize RR (1988) Quantitative immunocytochemistry using an image analyzer. II. Concentration standards for transmitter immunocytochemistry. J Neurosci Methods 26:25–34
Nunez DJ, Davenport AP, Emson PC, Brown MJ (1989) A quantitative “in situ” hybridization method using computer-assisted image analysis. Biochem J 263:121–127
Rentrop M, Knapp B, Winter H, Schweizer J (1986) Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNA's to frozen tissue sections under varying fixation and pretreatment conditions. Histochem J 18:271–276
Rogers KV, Vician L, Steiner A, Clifton DK (1987) Reduced preprosomatostatin messenger ribonucleic acid in the periventricular nucleus of hypophysectomized rats determined by quantitative in situ hybridization. Endocrinology 121:90–93
Rogers WT, Schwaber JS, Lewis ME (1987) Quantitation of cellular resolution in situ hybridization histochemistry in brain by image analysis. Neurosci Letters 82:315–320
Romano GJ, Shivers BD, Harlan RE, Howells RD, Pfaff DW (1987) Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization. Mol Brain Res 2:33–41
Segu L, Rage P, Boulenguez P (1990) A new system for computer-assisted quantitative receptor autoradiography. J Neurosci Methods 31:197–205
Schacther BS, Johnson LK, Baxter JD, Roberts JL (1982) Differential regulation of glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary. Endocrinology 110:1442–1444
Siperstein ER, Miller KJ (1973) Hypertrophy of the ACTH-producing cell following adrenalectomy: a quantitative electron microscopic study. Endocrinology 93:1257–1268
Stolz W, Scharffetter K, Abmayr W, Köditz W, Krieg T (1989) Automatic analysis method for in situ hybridization using high-resolution image analysis. Arch Dermatol Res 281:336–341
Swillens S, Cochaux P, Lecocq R (1989) A pitfall in the computer-aided quantitation of autoradiograms. TIBS 14:440–441
Taniguchi Y, Shiino M (1990) Immunoelectron-microscopic and morphometric study of the rat corticotrophs after adrenalectomy. Acta Anat (Basel) 137:45–48
Young III WS, Mezey E, Siegel RE (1986) Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry. Mol Brain Res 1:231–241
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Larsson, L.I., Traasdahl, B. & Hougaard, D.M. Quantitative non-radioactive in situ hybridization. Model studies and studies on pituitary proopiomelanocortin cells after adrenalectomy. Histochemistry 95, 209–215 (1991). https://doi.org/10.1007/BF00266769
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DOI: https://doi.org/10.1007/BF00266769