Alkaline fixation drastically improves the signal of in situ hybridization

Abstract

In situ hybridization (ISH) is widely used to detect DNA and RNA sequences within the cell and tissue sections. The important step in performing this technique is tissue fixation. We investigated the influence of the pH of the fixative on the outcome of ISH. Our studies indicate that alkaline formaldehyde dramatically increases the ISH signal with RNA probes. The increase in signal was observed for detection of low as well as for high abundance messages. The sensitivity of the method was increased 5- to 6-fold.

INTRODUCTION

In situ hybridization (ISH) is a powerful technique which enables visualization of specific nucleic acid sequences in morphologically preserved chromosomes, cells, tissue sections or whole embryos. ISH to mRNA allows the analysis of the spatial and temporal patterns of gene expression in physiological and pathological conditions, as well as during development. This technique provides an extra dimension to data obtained by nothern blot analysis or RNase protection assay, since it can resolve expression of transcripts in a single cell.

ISH was originally described by John et al. (1) and Gall and Pardue (2) for the detection of ribosomal gene sequences in cells using a radiolabeled RNA probe. Subsequently, a variety of techniques for cellular localization of both DNA and mRNA within tissue sections were developed using radioactive and non-radioactive probes. The non-radioactive ISH methods have become more popular due to safety concerns and they were recently demonstrated to be equally sensitive as radioactive ones (3). The non-radioactive methods appear particularly useful in studying the subcellular localization of mRNAs since they provide a much higher spatial resolution of the label than that obtained with radioactive methods (4). Furthermore, they allow the detection of several different transcripts in one experiment, by the use of differently labeled probes.

Despite the numerous protocols for ISH reported in the literature, researchers performing this technique still encounter a number of difficulties, especially when working with low abundance RNAs. Although many different protocols have been devised for both radiolabeled and ‘cold’ probes, a careful examination of these approaches reveals that certain steps are common to all protocols and are crucial for a successful outcome. One of these critical steps is tissue fixation. Fixation should ideally prevent the loss of cellular RNAs during hybridization while preserving accessibility of the target RNA to the probe. The primary fixative of choice of most investigators is 4% neutral buffered formalin.

Tissue fixation by formaldehyde works by crosslinking amino groups, thereby preventing loss of the mRNA target. During hybridization, high temperature and formamide remove some of these crosslinks. This process promotes penetration of the probe, but may also lead to unwanted loss of the target RNA. Thus, the ratio between the temperature of hybridization and the strength of fixation is very important to obtain an optimal signal. Furthermore, when using RNA probes the hybridization temperature should be high enough to ensure specific binding of the probe. In this study we investigated the effect of fixation conditions on the outcome of ISH and found that fixation of the tissue under alkaline pH dramatically improves the signal when using RNA probes.

MATERIALS AND METHODS

To prepare digoxigenin (DIG)-labeled cRNA probes for ISH, cDNA fragments of the genes of interest were subcloned in the vector pGEM9Zf (Promega) such that the resulting probes contained no polylinker sequences, as these have been shown to cause background signal during ISH (5). The probes were synthesized using in vitro transcription with a DIG RNA labeling kit (Boehringer Mannheim) and visualized on agarose gels. Probes were quantitated by spotting on nylon membrane and detected using anti-DIG antibodies conjugated with alkaline phosphatase. For detection of actin we used a 120 base long RNA probe, corresponding to the last 39 amino acids of mouse β-actin. For detection of the human zinc finger transcription factor ZAC we used a 294 base long RNA probe, corresponding to nt 1889–2182 of the human ZAC cDNA (GenBank accession no. AJ006354).

Our method is a modified version of the protocol of Scharen-Wiemers and Gerfin-Moser (6). We performed ISH with sections of tissues. Samples were taken on ice after surgery, quickly snap frozen in isopentane and stored for up to 6 months at –80°C. Tissue sections of 10–15 µm were cut, placed on Superfrost Plus slides (Menzel-Glaser) and air dried for 20–30 min at room temperature.

Tissue sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 9.5, for 60 min at room temperature (unless otherwise specified), washed twice with PBS for 3 min each and permeabilized with 1% Triton X-100 in PBS for 20 min at room temperature or with 70% ethanol overnight at 4°C. Subsequently, slides were washed three times with PBS for 5 min each and soaked in 5× SSC, 50% formamide for 15 min.

An aliquot of 100 µl of the hybridization solution (50% formamide, 5× SSC, 0.02% RNase-free BSA, 250 µg/ml bakers yeast RNA, 10% dextran), containing 200–400 ng/ml DIG-labeled RNA probe, was placed on the tissue sections. RNA probes were heated to 80°C for 1 min and iced before adding to the solution. The slides were coverslipped and placed in a humidified (5× SSC, 50% formamide) chamber to hybridize overnight at 55°C.

The next day the slides were washed in Coplin jars with 2× SSC, 50% formamide for 15 min at 55°C to remove coverslips. They were then washed in 2× SSC, 50% formamide at 55°C for 30 min once and twice for 30 min at 55°C in 0.2× SSC, 50% formamide.

After washing, the slides were transferred to 0.2× SSC at room temperature for 5 min and subsequently to buffer B1 (0.1 M Tris pH 7.5, 0.15 M NaCl) at room temperature for 5 min. The sections were then blocked by incubation in buffer B1 with 10% heat-inactivated sheep serum. After 1 h at room temperature the blocking solution was replaced by alkaline phosphatase-conjugated anti-DIG antibodies (Boehringer) diluted 1:5000 in B1 containing 1% sheep serum. The slides were placed in a humidified chamber overnight at 4°C; for high abundance RNA a 1 h incubation at room temperature can be sufficient, but overnight incubation greatly enhances the signal and reduces the color reaction time.

The following day the slides were rinsed three times for 5 min in buffer B1, equilibrated with buffer B3 (0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl₂) and placed in Coplin jars with buffer B3 containing substrates for alkaline phosphatase: 0.33 mg/ml nitroblue tetrazolium, 0.165 mg/ml bromochloroindolyl phosphate and 2 mM levamisole. The alkaline phosphatase color reaction was monitored by light microscopy. The reaction was stopped when desired (up to 3 days for low abundance messages) by placing the slides in TE, pH 8. The slides were then rinsed with water, excess liquid was removed and they were mounted in glycerol mounting medium containing DAPI or propidium iodate as counterstain.

RESULTS AND DISCUSSION

To investigate the influence of tissue fixation on ISH, slides with sections of human mammary gland were fixed in acidic (containing 10% acetic acid), neutral or alkaline (pH 9.5) 4% formaldehyde for 10 or 30 min and hybridized with a probe for β-actin. When samples were fixed for only 10 min, no signal was seen using acidic or neutral fixation, but a weak signal was observed following alkaline fixation (overnight color reaction; data not shown). Following 30 min fixation, a much stronger signal was detected using alkaline-fixed tissue (color appeared after 3 h; Fig. 1c), while a very weak signal was observed following neutral fixation (Fig. 1b). Again, no signal was observed after acidic fixation (Fig. 1a).

Figure 1.

Alkaline fixation improves detection of actin mRNA. Sections of normal human mammary gland were hybridized with DIG-labeled actin probe, using different conditions of fixation. The tissue sections were fixed for: (a) 30 min in 4% formaldehyde, PBS, 10% acetic acid; (b) 30 min in 4% formaldehyde, PBS, pH 7; (c) 30 min in 4% formaldehyde, PBS, pH 9.5; (d) 1 h in 4% formaldehyde, PBS, pH 9; (e) 1 h in 4% formaldehyde, PBS, pH 10; (f) 1 h in 4% formaldehyde, PBS, pH 12. The labeling in the epithelial cells is shown by the arrows. Sections (a)–(c) were developed overnight in alkaline phosphatase color reaction solution; sections (d)–(f) were developed for 6 h. The field is 1.7 × 1.2 mm.

We then studied alkaline fixation in more detail. Tissues were fixed for 30 or 60 min with 4% formaldehyde at pH 8, 9, 10, 11 or 12 and hybridized with the actin probe as before. We found that the strongest signal was obtained after fixation at pH 9 or 10 for 1 h (Fig. 1d and e). In this material, signal began to appear after 1 h of color reaction and was strong after 3 h. In tissue fixed at pH 11 and 12 the signal was reduced and even absent in some places (Fig. 1f).

We then went on to test our fixation conditions for detection of low abundance RNAs. ISH was performed on sections of human mammary gland using an RNA probe for the human zinc finger transcription factor ZAC, a low abundance message (7,8). After 1 h fixation in 4% paraformaldehyde at pH 9.5, the signal began to appear after 6–8 h and was abundant after overnight color reaction (Fig. 2c). No signal was observed in controls using an antisense probe (Fig. 2d) or with an irrelevant LacZ probe (data not shown). In contrast, ISH after 1 h fixation with neutral pH gave no signal following overnight color reaction. A weak signal appeared after 3 days of reaction (data not shown).

Figure 2.

Detection of ZAC mRNA by ISH, using alkaline fixation. Sections of cell pellets and normal human mammary gland were hybridized with the probe for human transcription factor ZAC. (a) Cal51 cells; (b) SaOS₂ cells; (c and d) normal human mammary gland. (a)–(c) Hybridization with the ZAC antisense probe; (d) hybridization with the ZAC sense probe. The signal is shown by arrows. All the sections were fixed for 1 h in 4% formaldehyde, PBS, pH 9.5, and developed overnight in alkaline phosphatase color reaction solution. The field is 1 × 0.7 mm.

To confirm the specificity of the ZAC probe, we performed ISH on cells which express ZAC, using non-expressing cells as a control (cell lines Cal51 and SaOS₂, respectively). Cells were trypsinized, pelleted and frozen in isopentane. The pellets were then cut into frozen sections and processed as above for ISH with a ZAC probe. As expected, we observed a positive ZAC signal in the cytoplasm of Cal51 ZAC-expressing cells (Fig. 2a) and no signal in SaOS₂ cells, even after prolonged incubation with the alkaline phosphatase substrates (Fig. 2b).

To investigate the ability of our new protocol to correctly detect the distribution of RNAs at the subcellular level, we performed control ISH with a fluorescently labeled oligo(T) probe. For this experiment, SaOS₂ cells were grown on coverslips and fixed for 1 h under neutral or alkaline pH. They were then processed for RNA ISH exactly as above but without addition of probe. After post-hybridization washing, cells were hybridized with oligo(T) (9). We observed that the signal had the same distribution after both types of fixation. As expected, it was diffusely present throughout the cytoplasm, while in the nucleus it had the characteristic ‘speckled’ pattern and was excluded from nucleoli (Fig. 3a and b). Thus, alkaline fixation did not alter the distribution of the cellular RNAs. Furthermore, in both cases the oligo(T) probe gave us a signal of similar intensity. This suggests that alkaline fixation does not improve retention of RNA during ISH, but rather increases accessibility of the target for the RNA probe, perhaps by facilitating denaturation of RNA and proteins during fixation at high pH. Subsequent treatment of the tissue with Triton X-100 or 70% ethanol helps to achieve good penetration of the RNA probe. Combination of a high temperature of hybridization and alkaline fixation results in a strong signal with minimal background.

Figure 3.

ISH of SaOS₂ cells with Cy3-labeled oligo(T) probe. Cells were fixed for 1 h in 4% formaldehyde, PBS: (a) pH 7; (b) pH 9.5. Following fixation cells were processed for RNA ISH and subsequently hybridized with Cy3-labeled oligo(T) probe. The field is 85 × 136 µM.

In conclusion, alkaline fixation significantly increased the signal of ISH. It gives a 5- to 6-fold reduction in color reaction time for abundant messages, while for low abundance RNAs the use of alkaline fixation allowed visualization of the signal, which was very weak or absent under fixation at neutral pH.

The protocol we have described is easy, reliable and gives good resolution. It works efficiently on sections of human breast, skin, placenta, mouse tissues, cells grown on coverslips and sections of cell pellets, for detection of both high and low abundance messages. Fixation under alkaline pH gives a significant increase in ISH signal for detection of cytoplasmic mRNA and can be applied to any ISH protocol, using RNA probes and paraformaldehyde as fixative.