Histomorphological Phenotyping of the Adult Mouse Brain

Anna Mikhaleva1, Meghna Kannan2, Christel Wagner2, Binnaz Yalcin2

1 Center for Integrative Genomics, University of Lausanne, Lausanne, 2 University of Strasbourg, Illkirch
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.12
Online Posting Date:  September, 2016
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This article describes a series of standard operating procedures for morphological phenotyping of the mouse brain using basic histology. Many histological studies of the mouse brain use qualitative approaches based on what the human eye can detect. Consequently, some phenotypic information may be missed. Here we describe a quantitative approach for the assessment of brain morphology that is simple and robust. A total of 78 measurements are made throughout the brain at specific and well‐defined regions, including the cortex, the hippocampus, and the cerebellum. Experimental design and timeline considerations, including strain background effects, the importance of sectioning quality, measurement variability, and efforts to correct human errors are discussed. © 2016 by John Wiley & Sons, Inc.

Keywords: brain morphology; histology; mouse; phenotyping

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Table of Contents

  • Introduction
  • Basic Protocol 1: Brain Fixation and Trimming
  • Basic Protocol 2: Brain Sectioning
  • Basic Protocol 3: Luxol‐Nissl Staining
  • Basic Protocol 4: Whole‐Slide Scanning
  • Basic Protocol 5: Data Collection and Data Quality Control
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Brain Fixation and Trimming

  • Histological cassettes, macro (VWR Collection, cat. no. 720‐2206) and small (VWR Collection, cat. no. 720‐2192)
  • Male (or female) mouse, 16 weeks of age (or any other postnatal age)
  • 10% (v/v) neutral buffered formalin, Tissue‐Tek (Sakura, cat. no. 8726)
  • 70% (v/v) ethanol
  • Laboratory precision balance (Ohaus 092567, Dominique Dutscher)
  • Dissection instruments: surgical scissors (Fine Science Tools GmbH, cat. no. 37500‐00), surgical blade (Braun Medical AS, cat. no. 16600584), pair of extra‐thin straight scissors (Fine Science Tools GmbH, cat. no. 14088‐10), short and long forceps (Fine Science Tools GmbH, cat. no. 11063‐07 and 11693)
  • Plastic petri dish (Falcon, cat. no. 353003)
  • 500‐ml capped jars (Verlabo 2000, cat. no. 520050)
  • Fume hood
  • Inkjet printer for tissue cassette (Leica IP S)
  • Microtome blade (Dura Edge Inc., cat. no. 7223)
  • Tissue processor and accessories (Sakura Tissue‐Tek VIP)
  • Histology embedding molds, stainless steel (Leica EG 1140 H)
  • Cold plate for modular tissue embedding system (Leica EG 1150 C)
  • Storage racks for paraffin blocks (VWR International SAS, cat. no. 631‐1050)
CAUTION: Formalin is highly hazardous and flammable. Avoid skin and eye contact, inhalation, or ingestion. In case of contact with eyes or skin, rinse immediately with plenty of water and seek medical advice. In case of inhalation, move person to fresh air space.

Basic Protocol 2: Brain Sectioning

  • Formalin‐fixed paraffin‐embedded tissue (prepared in protocol 1)
  • Gelatinized water (see recipe)
  • SUPERFROST histology glass slides (Thermo Scientific, cat. no. ISO 8037/1)
  • Inkjet printer for microscope slide (Leica IP S)
  • Paraffin trimmer (Leica Surgipath Trimease)
  • Water bath (GFL 1052)
  • Microtome Leica RM 2145
  • Microtome blades (Dura Edge Inc., cat. no. 7223)
  • Pair of forceps (Bochem, cat. no. 1132)
  • Fine paintbrush (Rein Rotmarder no. 3)
  • Light microscope (Leica DM1000)
  • 100‐place histology slide boxes
  • Incubator

Basic Protocol 3: Luxol‐Nissl Staining

  • Sections mounted on histological slides (from protocol 2)
  • Xylenes (VWR International AG, cat. no. 28073.328)
  • 100%, 90%, and 70% (v/v) ethanol
  • 0.1% (w/v) Luxol fast blue (see recipe)
  • 0.1% (w/v) cresyl violet acetate (see recipe)
  • 0.05% (w/v) lithium carbonate (see recipe)
  • Staining racks (Electron Microscope Sciences, cat. no. 70321‐10)
  • Fume hood
  • Staining glass dishes with lids (Electron Microscope Sciences, cat. no. 71424‐DL)
  • Hot plate (Medite, TFP40)
  • Leica automated staining unit (SAKURA Tissue‐Tek Prisma & Film)
  • Rectangular 20‐slide staining dishes unit with glass covers (Electron Microscope Sciences, cat. no. 70312‐20)
  • Aluminum foil
  • Shaker (GFL 3005)
  • Water bath (GFL 1002)
  • Filter paper (VWR International SAS, cat. no. 5160838)
  • Pertex (Microm France, cat. no F/00811)
  • Coverslipper (Leica CV5030)
  • Storage racks for microscope slide (VWR International SAS, cat. no. 631‐1069)
CAUTION: Xylenes are highly toxic and flammable. Avoid skin and eye contact, inhalation, and ingestion. In case of contact with eyes or skin, rinse immediately with plenty of water and seek medical advice. In case of inhalation, move person to fresh air space.

Basic Protocol 4: Whole‐Slide Scanning

  • Whole slides to be scanned (from protocol 3)
  • Light microscope (Nikon SMZ800)
  • Permanent marker
  • Tissue wipes (Kimtech, cat. no. 34120)
  • Slide scanner (Hamamatsu, NanoZoomer 2.0HT, C9600 series) and accessories (racks and NanoZoomer digital pathology, version 2.5.64 software)

Basic Protocol 5: Data Collection and Data Quality Control

  • Computer with the following specifications: Windows (64‐bit) operating system, 8 MHz processor, 16 GB RAM, 4 TB hard drive, and AMD Radeon HD 7450 DP 1st graphic card
  • ImageJ software (http://imagej.nih.gov/ij/).
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