Nonradioactive in Situ Hybridization Histochemistry in Leukemic and Nonleukemic Culture

Abstract

Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HCl followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization.

High-resolution In Situ Hybridization and TUNEL Staining with Free-floating Brain Sections

“…The nonradioactive in situ hybridization protocol used for tissue sections is essentially similar to the nonradioactive in situ hybridizaton procedure used on our glial cell cultures (Maki et al 1997;Granneman et al 1998). We found that labeling small amounts of probe gave the most intense staining of cells, and our at- The neuroepithelial layer is thickened at the dorsolateral border.…”

“…No staining was found in the control tissue. Our previous in situ hybridiztion studies (Maki et al 1997;Granneman et al 1998) with pBR322 DIG-labeled cDNA produced no signal using a similar procedure, and RNAse treatment also abolished the signal.…”