ch 15

• Mounting is the last step in tissue processing that results in a permanent histological preparation suitable for microscopy, after adhesion of the sections on to the slide and appropriate staining of the tissue.
• After cutting, sections are floated out on a water-bath. Bubbles accumulating under the ribbon may be removed with a smooth teasing needle, care being taken not to tear the section. Bubbles may also be removed by pulling the ribbon very gently across the long edge of a glass slide held below the section in the water bath.
• After draining, the sections are fixed to the slides. This can be done either by leaving the slides in a 37°C incubator overnight, by placing the slides in a wax oven at 56° to 60°C for 2 hours, or by drying the slides on a hot plate at 45° to 55°C for 30 to 45 minutes. For more delicate tissues like the CNS tissue or brain, a longer drying time at lower temperature (e.g. 37°C for 24 hours or longer) is recommended to avoid splitting and cracking of the section due to excess heat.
• Another alternative to drying is by the use of adhesives. To promote adhesion of sections, adhesives may be spread thinly and evenly on a clean grease-free slide which is then gently approximated to the end of the ribbon, and drawn upwards in a near vertical motion.
• An adhesive is a substance which can be smeared on to the slides so that the sections stick well to the slides. The choice of slide and adhesive will be influenced by the staining methods to be subsequently applied. Slides must always be grease- and dust- free and stored and handled correctly.
• If staining is to include antigen retrieval (IHC), enzyme pretreatment for in-situ hybridization (ISH), or prolonged incubation steps, charged slides or an adhesive must be used.
• Some special stains, particularly those that employ alkaline reagents, can also cause sections to lift
• Slides must always be accurately and appropriately labelled in a manner compliant with local regulations.
• Adhesives are not necessary for routine staining, provided that the slides are clean and free from grease. However, they are essential for methods that require exposure of sections to acids and alkalis (especially ammoniacal silver solutions) during staining.
• If clean grease-free slides are used and sections are adequately dried, the sections will not float off during staining and adhesive will not be necessary.
• The most commonly use adhesive is Albumin.
• There are still certain instances when sections may float from the slide:

  (1) For urgent cryostat sections to be submitted for immunocytochemistry

  (2)     For central nervous system tissues

  (3)     For tissues containing blood clot

  (4)     For tissues which have been decalcified

  (5)     When sections are to be subjected to high temperatures.
• One disadvantage of using albumin is that it retains some of the stain and gives a dirty background.
• Thymol resistant organisms growing in the adhesive (albumin) have been known to contaminate gram-stained sections and cause confusion during microscopic examination.
• Poly-L-lysine, also a favorite adhesive, can be bought as a 0.1 % solution and further diluted (1 in 10 with distilled water) when ready to use. Sections are coated with this dilute poly-L-lysine and allowed to dry.
• With time, the adhesive ability of Poly-L-lysine slowly loses its effectiveness. Therefore, the coated slides should be used within a few days.
• Aminopropyltriethoxysilane (APES) is a better section adhesive and coated slides can be stored for a long time.
• APES is invaluable in cytology particularly for cytosine preparation of proteinaceous or bloody material.
• Mayer's egg albumin is the most commonly used because it is very easy to make, is convenient, and is relatively inexpensive.
• A drop of Mayer's egg albumin is usually smeared into the clean glass slide before sections are oriented.
• Sections which have been creased on cutting may be stretched by gentle heating before attaching them into slides.
• During staining, the excess of albumin may also take up the stain and interfere with diagnosis; hence, it should be wiped off from the slide to remove any excessive solution
• For celloidin sections, egg albumin is smeared on the slide. The section is then transferred from 95% alcohol bath to the slide, pressed flat on the slide with a smooth filter paper coated with thin celloidin mixture.
• Dried Albumin - dried, and stored in 70% alcohol until it is ready for staining.
• crystals of thymol to prevent the growth of molds
• Adding up to 30 ml of 1% aqueous gelatin to the water in a floating out bath and mixing it well is a most convenient alternative to direct coating of slides.
• Gelatin-formaldehyde mixture

  Allow coated slides to dry at 37°C for one hour or overnight before use.
• Poly-L-Lysine

  This aqueous detergent can be purchased as a 0.1% solution which is further diluted 1:10 with distilled water (final dilution to 0.01%) prior to use. Sections are coated with this diluted poly-L-lysine and allowed to dry.
• Poly-L-Lysine


  This is widely used as a section adhesive in immunohistochemistry.
• PolyL-lysine coated slides must be used within a few days after they are prepared, since its effectiveness as an adhesive slowly decreases in time.
• APES-coated slides are better than poly-L-lysine coated slides because they can be stored for a long time without losing their adhesiveness.
• APES-coated slides are very useful in cytology, particularly for cytospin preparations of proteinaceous or bloody material.