Cryosections probes

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

For cryosectioning, tissue samples are quickly frozen with or without freezeembedding medium (e.g., Tissue Tek Miles Laboratories) and stored at 80°C until analysed. Optionally, aldehyde prefixation can also be used for tissue and organ probes before snap-freezing. Cutting of frozen tissue blocks is performed with a cryostat (a microtome mounted in a freezing cabinet). 

A microwave oven equipped with Pello 3420 Load Cooler attachment is set at 25°C and full power. Two beakers, each filled with 500 ml of water, are placed in previously determined hot spots in the microwave oven (see pages 102-103). Grids containing the thin cryosections are floated (section side down) sequentially on small drops of 0.15% glycine and 1% BSA for 15 sec and 5 min, respectively. The reagent drops are placed on a clean disposable plastic surface which has been placed on the cold spot in the oven (an area between the two beakers of water). The local temperature is controlled using the microwave temperature probe immersed in a tube of water placed close to the grids. 

The hybridization and washing conditions are identical for both paraffin sections and cryosections. However, different steps of treatments prior to hybridization have to be performed for each type of sections (described, respectively, in Subheadings 3.4.3. and 3.5.3.). In the case of paraffin sections, the pretreatments consist of an incubation in an organic solvent to remove the paraffin wax, followed by a mild protease treatment to improve the penetration of probe, and an acetylation step that reduces nonspecific electrostatic binding of the negatively charged probe. In the case of ciyosections, the tissues are postfixed sequentially in acetone and formaldehyde and acetylated as for paraffin sections. 

Most of the published studies have used probes which detect all mRNA isoforms of a given RAR or RXR. It is also possible to generate isoform-specific riboprobes by limiting the template DNA to the specifically spliced region. In situ detection of individual RAR (RXR) isoforms poses problems, however, because of limitation in the nucleotide length of isoform-specific probes (e.g., 400-500 nt in the case of RAR isoforms), and in the very low abundance of individual mRNA isoforms Nevertheless, there have been recent reports of ISH detection of RARP isoforms in chick embryos (17,18) and RARy isoforms can be specifically detected on cryosections of mouse embryos (unpublished data) Detailed procedures for cloning, amplification, and purification of plasmid DNA, which are beyond the scope of this chapter, can be found in refs. 20 and 21... 

---