Subbed slides

Fix the cell bearing coverslips to slides as above. The use of subbed slides is unnecessary. 

Transfer ribbons of sections into 0.2 X subbing solution on subbed slides. Place on a slide warmer at = 45 C to stretch the sections, remove excess subbing solution and dry at 42°C for 24 h. 

After 15 min, trim the block, section the block and collect sections onto warm subbed slides or coverslips coated with Histostik (Accurate Chemical and Scientific, Co.) with standard methods. 

Cut sections (5-20 (tm) of unfixed tissue in a cabinet cryostat (-20 C ) directly onto subbed slides. Allow the sections to air dry overnight at room temperature in a dark, dust-free place. Alternatively, sections on slides may be stored at -TO C to -80°C (2-3 mo) in a sealed plastic slide box. Stored slides should be allowed to warm to room temperature before use. 

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... 

Stir each emulsion with clean glass rods and dip a clean subbed slide up and down ( 50 times) to remove air bubbles. Check for a homogeneous coating of emulsions by dipping clean subbed slide in each respective emulsion. 

It is not necessary to use subbed slides in this procedure because the roots have not been treated with enzymes that may prevent adherence of the cells to the slide. 

OCT (TissueTek) and gelatin-subbed slides or Superfrost Plus slides (Fisher). 

SUBBED SLIDE WITH RECEPTOR LABELED TISSUE... 

Subbed slides dip racks of microscope slides into a mixture of 1% gelatin plus 1% formalin in distilled water drain the slides and dry in a warm oven... 

Thaw the drop and touch onto a subbed slide. 

Sections are floated on a clean water bath filled with distilled water at a temperature slightly below the melting point of the wax. After the sections spread out, subbed slides are placed beneath the floating ribbons to collect them and dried on a 37°C heating plate for 1 h, and then overnight in a 37°C oven. 

Wash slides, dip in subbing solution, and allow to dry in a dust-free place. These slides may be stored indefinitely in a dry, dust-free container. Subbed slides give a better retention of cytological preparations. 

Invert a subbed slide over the coverslip and press it down lightly. 

Cut sections (10-14-pm thick) and transfer to slides (subbed slides are not necessary). ... 

Transfer sections to a subbed slide (see Protocol 13.4 Notes, below) by rolling sections first onto a beveled wooden stick and then off of the stick into a puddle of H O on the subbed slide. 

Subbed slides are not absolutely necessary, but because the subbed surface increases the surface tension of H O, the drop of H O forms a bead rather than spreading out in a thin film across the slide. This is convenient because the sections are consequently confined to a smaller surface area on the slide. 

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