Tissue chopper

Preparation of brain slices is an indispensable procedure for a variety of experiments. The goal of the slice preparation is to obtain a thin piece of brain tissue containing the cells of interest and to maintain the slice in a viable (although artificial) condition that is similar to its in vivo environment. In this chapter we describe procedures that are fundamental for brain slice preparation. Because the hippocampus is the most widely used tissue for brain slices, Its isolation will be used here to illustrate the steps of the procedure. Two slicing methods, using either a vibratome or a tissue chopper, are described. Our discussion of these methods covers the steps from the decapitation of the animal to the storage of slices in artificial cerebrospinal fluid (aCSF). Some of the... 

The vibratome and tissue chopper methods are two major techniques used to slice tissue blocks. The vibratome seems to produce better slices and is gaining popularity. The available commercial vibratomes include the Vibratome series from Technical Products International (St. Louis, MO), and the Vibroslice... 

The tissue chopper, on the other hand, has the advantage of low cost and faster slicing. The available commercial tissue choppers include the Tissue Sheer from Stoelting (Wood Dale, IL) and Mcllwain Tissue Chopper (Mickle Laboratories Engineering, Gromshall, Surrey, UK), Slicing is improved if the subarachnoid membrane is removed. 

Tissue chopper apparatus (Stoelting Tissue Sheer)... 

Some authors isolate the hippocampus in a way as described in the tissue chopper method. The middle portion of the hippocampus is cut out and mounted onto the vibratome slicing stage with cyanoacrylate glue In our opinion, the tissue block method described in this chapter is superior, since it reduces the direct handling of the hippocampus. 

Using a sterile, wide-bore, plastic transfer pipet, transfer the hippocampi to a polyester film immobilized on the stage of a Mclllwam tissue chopper (Bnnkman, Westbury, NJ see Note 7). 

Wipe all tissue culture incubators, hoods, tissue choppers, and microscope stages with 70% ethanol on a regular basis. It is advisable to have a dedicated tissue chopper that remains in a sterile tissue culture hood. Most contamination problems occur during the preparation of the slice cultures. Contaminants are generally of two types yeast and bacteria. Control bacteria by adding chloramphenicol... 

The hippocampus is sliced on a 200 pm polyester film of 2 x 3 cm (Pearl Paint) that has been extensively washed with 70% ethanol and air dned in a sterile hood Any plastic film can be used to support the hippocampus The film must be sterile and must resist being sliced by the tissue chopper blade There are consistent differences between cultures of the septal and temporal hippocampus that likely result from developmental gradients in the immature hippocampus (17), At postnatal d 10-11, the temporal end of the hippocampus is more mature than the septal end. Therefore, consistent differences can be observed between slice cultures originating from the temporal end or septal end. In particular, the mossy fiber projection differs between septal and temporal cultures (17) In septal cultures, there is mossy fiber sprouting mossy fiber terminals are found in the inner molecular layer of the dentate gyrus and the CA3 pyramidal cell layer. In temporal cultures, the mossy fiber projection is normal. This likely results in differences in slice culture excitability since epileptiform activity is more readily induced in septal cultures than temporal cultures (17). 

Mcllwain tissue chopper (Mickle Laboratory Engineering Co., Ltd.). 

Cut 300 pm transverse slices using a Mcllwain tissue chopper (see Note 20). 

The quality of the slice depends to a certain extent on the method used to prepare the slices (40,41). Using the Mcllwain tissue chopper it is possible to prepare lOO-pm thick slices. These are tedious to work with. However, slices of 300- jm thickness can, with some care, be bandied routinely. Furthermore, 300- am presents a thickness that is acceptable in terms of oxygen supply to the deeper layers of the slice (42,43). 

Mcllwain tissue chopper (Brinkmann Instruments, Westbury, NY). Sterile 35-mm petri dishes. 

Cut 350 pm-thick parasagittal slices of cerebellum with a tissue chopper. SrrNote 12. 

Liquid samples such as milk do not normally require application of any pretreatment procedure. Semisolid samples such as muscle, liver, and fat tissues usually require more intensive sample pretreatment for tissue break-up. The most popular approach is grinding the sample in a food chopper or homogenization in a Waring blender to expose residues to the extraction solvent. Fatty tissue samples are usually warmed at 35 C until fat melts (491-493), or sometimes blended with immersion blender (494). A fat sample that has been blended with immersion blender melts to produce yellow oil, whereas oil does not separate... 

Combine 60 g washed endosperm tissue with 90 ml chilled grinding medium and chop vigorously in an onion chopper for 6 minutes. 

The double-isotope dansyl microassay has also been used to measure release of amino acids from brain preparations in vitro (Snodgrass and Iversen, 1973). Rat cortical tissue was chopped using a Mcllwam chopper and suspended in Krebs-bi-carbonate-glucose medium. Electrical stimulation was applied and, following superfusion, the medium was changed for a Krebs-bicarbonate buffer containing 55 mM KCl. In this way,... 

Before analysis, meat samples must be groimd in a suitable mincer or bowl-cutter to produce a homogeneous sample. Connective tissue is a particularly difficult component of the meat sample to homogenize and this is of importance in collagen analysis. Suitable comminution of meat samples is specified as a mincer (meat chopper) plate size of 4 mm or less. 

Figure 4.5 Top Scheme of a two-chopper TR microscope. Redrawn from Reference [38], Bottom Prompt (a) and delayed (b) red luminescence (>590 nm) of an AO-stained polytene chromosome covered with tissue (excitation wavelength Ar laser, 488 nm). Reproduced with permission from [38], Copyright 1991, Elsevier...

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