Observations on the simultaneous use of digoxigenin- and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry

In situ hybridization: Methods and applications

“…Two major methods for labeling and detection are used: (a) radioisotope labeling, such as 3 H, 35 S, 125 I, 32 P, and recently 33 P, which is detected with x-ray film and/or by emulsion autoradiography; and (b) nonisotope labeling, such as biotin, digoxigenin, fluorescein, alkaline phosphatase, or bromodeoxyuridine (BrdU), which is visualized by histochemistry or immunohistochemistry (IHC) detection systems.…”

“…35 S-labeled probes are most commonly used, representing the most sensitive and rapid results (typical exposures of about 1 week) compared to other isotopic labeling (3). The half-life of 35 S is 87 days, and labeled probes should be used within 1 month or so.…”

Neuronal colocalization of mRNAs for neurotrophins and their receptors in the developing central nervous system suggests a potential for autocrine interactions.

“…Slides were first processed for the alkaline phosphatase-linked color reaction (for the detection of the digoxigenin-labeled probe) and then coated with autoradiographic emulsion (Ilford, L4 or K5) and processed for the detection of the 35S-labeled oligonucleotide probe. Ilford emulsion was used in order to avoid positive chemography induced by the alkaline phosphatase product (37). Hybridization with 35S-labeled probes was considered specific where the accumulation of exposed silver grains exceeded 5 times background (38).…”

Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats.

“…Therefore, use of the Parlodion treatment before autoradiography was required in both instances. As an alternative to Parlodion coating, chemography can be eliminated by using Ilford K-5 instead of the Kodak NTB3 nuclear track emulsion to visualize autoradiographic signals in BCIP-NBT-labeled sections (39). This approach, however, does not prevent solubilization oft!…”

“…This solution was replaced with 70% ethanol-300 mM ammonium acetate and the sections were mounted on gelatin-coated glass slides with a fine-bristled paintbrush and air-dried. Some mounted sections were further dehydrated through 100% ethanol, air-dried, then dipped into 1% Parlodion (Sigma) in acetone (39). After a short drying period, sections were coated two more times with Parlodion solution in the same way.…”