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Abstract

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Protocol

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Neuroscience

Simultaneous Cryosectioning of Multiple Rodent Brains

Published: September 18th, 2018

DOI:

10.3791/58513

1Department of Child Health, University of Arizona College of Medicine, 2Department of Biological Sciences, University of Bath, 3BARROW Neurological Institute at Phoenix Children's Hospital, 4Phoenix Veteran Affairs Healthcare System, 5Department of Basic Medical Sciences, University of Arizona College of Medicine

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present the protocol for a novel method, which we call Megabrain, developed to cryosection multiple rodent brains simultaneously for downstream immunohistochemical procedures. A Megabrain allows for the production of single slides containing tissue from multiple animals. This technique has been optimized to cut coronal sections from 9 adult rat hemispheres, or 5 adult full brains, simultaneously. Therefore, the technique is most applicable in large immunohistochemical studies or other analyses done on slide-mounted brain tissue from a large cohort of animals.

Immunohistochemistry involves the use of specific antibodies directed aga....

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The Megabrain technique has been optimized using whole and hemisected brains from male adult C57Bl6 mice5, juvenile Sprague-Dawley rats, and adult Sprague-Dawley rats that were transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde6. Similar outcomes can be accomplished in other strains of mouse and rat, in both sexes and at different ages. The study to generate data for this manuscript used juvenile Sprague-Dawley rats and was appro.......

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A positive end result to this procedure is tissue that lies flat on the slide, with no bubbles or tears, in the orientation in which they were frozen. Tissue sections are evenly spaced apart and easily identifiable due to good placement of the brain in the OCT and good notation as demonstrated in Figure 1. Assuming that the brain tissue was collected from animals of a similar age, and that the tissue was properly aligned in the OCT, sections collected on a sl.......

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It should be considered during this procedure that the temperature of the Megabrain and its surroundings must be constantly monitored to prevent thawing and re-freezing of the tissue. The brain can only be removed from the -20 °C freezer up to 3 times as every time it is touched and left in the cryostat, with a warmer fluctuating temperature, the tissue thaws and refreezes, causing a jelly like texture and abnormal tissue integrity10. Therefore, it is optimal to cut the Megabrain all at once........

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PCH Mission Support Funds supported the research reported in this manuscript. The authors would like to thank Daniel Griffiths for taking the images used in the figures.

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Name Company Catalog Number Comments
Andwin scientific tissue-tek CRYO-OCT compound (case of 12) Fisher Scientific 14-373-65
Thermo Scientific Shandon Peel-A-Way Disposable Embedding Molds Fisher Scientific 18-41
Fisherbrand High Precision Straight Broad Strong Point Tweezers/Forceps Fisher Scientific 12-000-128
Fisherbrand 20mL HDPE Scintillation Vials with Polypropylene Cap Fisher Scientific 03-337-23
Sucrose, poly bottle 2.5 kg Fisher Scientific S2-212 Both 15% and 30% sucrose concentrations need to be made up.
2-Methylbutane (Certified), Fisher Chemical Fisher Scientific O3551-4
PYREX Tall-Form Beakers Fisher Scientific 02-546E
Fisherbrand General Purpose Liquid-in-Glass Partial Immersion Thermometers (-50° to +50°C) Fisher Scientific 13-201-642
Fisherbrand Scoopula Spatula Fisher Scientific 14-357Q
STANLEY Razor Blade Grainger 4A807
Edge-Rite Microtome blades Fisher Scientific 14-070-60
Microscope slides (1" frost) - white Fisher Scientific 22-034-979
Gibco PBS (Phosphate Buffered Saline) 10X, pH 7.2 Fisher Scientific 70-013-032 Dilute to 1X  before use
15 piece fine paint brushes Amazon B079J12ZRV
PAP pen abcam ab2601
Chuck Shown in Figure 4 was custom made by a lab technician, however similar sizes are available to order from other companies commercially. Electron Microscopy Sciences EMS065

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