If non silane-coated microscope slides are used, the sections will float off during the hybridisation. 3-aminopropyltriethoxysilane - AES or APES - has been found to be the best tissue - to - slide adhesive for ISH with cRNA and DNA probes 2,3,4. Nearly all (> 99%) tissue sections are retained, even if the slides are heat-denatured at 95oC (eg as for DNA ISH)
** AES is extremely irritant - it must be used in a fume hood **
Glass microscope slides Menzel SuperFrost colour Objekttrager : boxes of 50 (76 mm x 26 mm) slides Deconex-90 Borer, Zuchwil (5 litres) 3-aminopropyltriethoxysilane Sigma - Number A3648 (100 ml) Acetone Analar grade eg from BDH (5 litres) Distilled water
Acetone-resistant slide racks 4 x Lipshaw metal slide staining racks, each holding 60 slides, or equivalent racks Acetone-resistant staining dishes 5 x Lipshaw glass staining dishes with a metal lid, to hold 60-slides metal racks, or equivalent dishes Dust-free plastic containers large enough to hold ~250 slides each
Load the glass slides into metal staining racks and clean by immersion in 2% Deconex-90 in distilled water. Rinse well in distilled water and leave to air dry in the racks
1 In a fume hood, make up a fresh 2% (v/v) solution of AES in acetone (~1 litre) using glassware 2 Rinse slides in an acetone bath 5 minutes 22oC 3 Coat slides in 2% AES-acetone 5 - 15 minutes 22oC 4 Rinse slides : Acetone 2 - 5 minutes 22oC Acetone 2 - 5 minutes 22oC Distilled water 2 - 5 minutes 22oC 5 Dry slides overnight 6 Store in a dust free container at room temperature The slides keep for ~ 6 months although they lose some of their adhesiveness with time
1 This protocol is a modified version of that of Maddox and Jenkins 2 Some glass slides contain significant amounts of a-particle emitting isotopes which will produce a visible background during liquid film autoradiography. Menzel SuperFrost Objekttrager have always performed well. They also have frosted ends which are easy to mark with pencil, which is insoluble in xylene and toluene. Pencil has the additional advantage of producing an autoradiographic image on X-ray film 3 A set of staining racks and dishes should be reserved for slide coating : the racks should not be use for routine histological staining in the interim 4 If the slides are dirty, the AES coating will be uneven and the distilled water will 'bead' on the slides after coating. The uneven AES coat decreases effective adhesion between the tissue and slide. The 'beads' are visible after hybridisation as dark rings on both the film and slide autoradiographs. The slides can also be cleaned by loading them into racks and putting them through a domestic / commercial dishwasher 5 AES solutions greater than 2% produce very 'sticky' slides, but these frequently have patchy, granular white deposits and are not suitable for autoradiography 6 Use glassware to make up the AES solutions unless you are very sure that the plastic is AES and acetone-resistant. 7 Fresh AES solution should be made each time slides are coated. 1 litre of 2% AES will coat ~750 slides. The solution will slowly turn yellow on standing. Dispose of used solution in a sealed glass Winchester bottle 8 The slides can be dried at either room temperature or at ~ 80oC. Drying at high temperature produces slides that are very 'sticky'. However, they do not seem to last as well as those dried at room temperature 9 AES slides do not retain liquid film emulsion as well as gelatine-chrome alum subbed slides. Consequently, very thin emulsion films (diluted > than 50:50 with water) may not be retained during development and staining 10 The AES-coating possibly generates a higher background than gelatine-chrome alum
2 P. H. Maddox and D. Jenkins (1987) 3-aminopropyltriethoxysilane (APES) : a new advance in section adhesion J Clin Pathol 40:1256 - 1260 3 G. Niedobitek and H. Herbst (1991) Applications of in situ hybridisation International Review of Experimental Pathology. Ed Richter, G.W.Solez, K. 32:1 - 56. Harcourt Brace Jovanovitch 4 M. Rentrop, B. Knap, H. Winter and J. Schweizer (1986) Aminoalkylsilane-treated slides as supports for in situ hybridisation of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions Histochem J 18:271 - 276