Riboprobes

Sense- or antisense-radiolabeled riboprobe (concentration adjusted to 0.5 X 105 to 5 X 105 cpm/section)... 

Fig. 4. PK2 transcript (DIG-PK2 antisense riboprobe) associated with infiltrating cells, possibly neutrophils and/or macrophages in rat paw, 12 h after CFA injection (B). Sense probe (C) or saline injection (A) did not generate specific signal.

FIG. 7. SNS Na channel is not present within the normal brain, but is expressed in cerebellar Purkinje cells within brains obtained at post-mortem from MS patients. Panels on left show in situ hybridization with SNS-specific antisense riboprobes, and demonstrate the absence of SNS mRNA in control cerebellum (c) and its presence in Purkinje cells in post-mortem tissue from two patients with MS (a, b). No signal is present following hybridization with sense riboprobe (d). Panels on right show immunostaining with an antibody directed against SNS, and illustrate absence of SNS protein in control cerebellum (g, arrow indicates Purkinje cell) and its presence in MS (e, f). Modified from Black et al (2000). 

A.D. Stiles and B. Moats-Staats noted in the Winter 1993 Red Book Bulletin that riboprobed Northerns can be routinely stripped and rehybridized (on GeneScreen nylon). The stripping solution contains 10 mM sodium phosphate (pH 6.8) in 50% formamide. The membrane is incubated in 100 ml for 1 h at 70°C, rinsed with 25 mM sodium phosphate, and submitted to autoradiography to check whether any signal... 

The human Y1 mRNA is about 3.5-4 kb and was detected in the neuroblastoma cell line SK-N-MC by Northern hybridization (Larhammar et al., 1992). An in situ hybridization study with a riboprobe reported widespread distribution in human fetal and adult organs, e.g. colon, kidney, heart, placenta and adrenal (Wharton etal., 1993). However, a Northern hybridization in the same study detected a single mRNA of only 2.2 kb suggesting that the specificity of the probe for Y1 mRNA may be questioned. Curiously, in the same study the size for NPY mRNA was reported as 3.3 kb, which disagrees with the size seen in a human pheochromocytoma (0.8 kb) as well as the size of the human cDNA clone which ends with a poly(A) tract (Minth et al., 1984). 

Key Words In situ hybridization nonisotopic digoxigenin mRNA riboprobe CBi colocalization emulsion autoradiography. 

The desired concentration is approx 2 pM digoxigenin-labeled riboprobe/ mL hybridization buffer (usually this works out to be 2-6 pL probe/50 pL buffer)... 

Calculate required quantities of materials (i.e., probes, hybridization buffer, etc.) based upon number of slides and cover slip length required to hybridize all shde-mounted sections in the assay. This can be easily automated in Excel. Riboprobe hybridization buffer must be prepared with at least 20% more volume than would be calculated otherwise based on the number of slides. 

Tissue sections are obtained from rats that are killed by decapitation. The brains (or other required tissue) are rapidly removed and frozen in isopentane that has been precooled (-30°C) on dry ice. Sections (14 pM) are cryostat-cut and mounted (two sections per slide) under conditions that minimize RNase contamination. Mounting sections close to the bottom of the subbed slides will conserve reagents required for double-label ISH. Multiple adjacent series of sections are obtained, depending on need. For example, it is advisable to take a series for each [ Sj-labeled riboprobe both alone and in combination with each appropriate digoxigenin-labeled riboprobe. Slide-mounted sections are sorted at -20°C (in cryostat or cold room) prior to the assay to obtain the required number of near adjacent series. Slides brought to room temperature cannot be... 

Slides are placed in hybridization trays containing Whatman filter paper soaked in standard saline citrate (SSC)/formamide solution. Riboprobe hybridization buffer is applied to tissue using a repeater pipet, and slides are cover slipped. The cocktail of digoxigenin and p S]-labeled ripobrobe hybridization buffer (or the [ Sj-labeled rihoprohe hybridization buffer alone) is appUed to tissue in fixed voiumes determined by the size of the cover slip required to cover the sections (i.e., 50 pL per 30-mm cover sUp for sections mounted one or two per slide or 100 oL per 5- mm cover sUp for sections mounted three or four per sUde). Slides are placed in a humidihed chamber and incubated overnight at 52-55°C. 

Prepare riboprobe hybridization buffer (ribo HB) mixture based on required quantities ... 

Develop film (Kodak MR film) to produce film image of cells labeled by the [ S]-labeled riboprobe (see Note 8). The film image is appropriate for quantifying mRNA densities using autoradiography and computer-assisted densitometry. 

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