Standardized Post‐Mortem Examination and Fixation Procedures for Mutant and Treated Mice

Cristina Antal1, Stéphanie Muller2, Olivia Wendling3, Yann Hérault3, Manuel Mark4

1 Institut d'Histologie, Faculté de Médecine, Strasbourg, France, 2 Mouse Clinical Institute, Illkirch, France, 3 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/Université Louis Pasteur, Illkirch, France, 4 Hôpital Universitaire de Strasbourg, Strasbourg, France
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo100118
Online Posting Date:  March, 2011
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Abstract

A procedure for post‐mortem examination (or necropsy) of mice is provided. The aim is to obtain a “holistic” picture of organs and systems at the anatomical and histological levels. The major issue is tissue preservation, which is achieved by rapid transfer into a fixative solution, usually neutral buffered formalin. Fixation is the first of the four basic steps in histopathological analyses of tissues, which also include embedding, sectioning, and staining. The protocols provided here describe routine methods for tissue fixation, as these methods are integral parts of any necropsy procedure. There is also a Strategic Planning section that addresses the overall approach to histopathological evaluation, as well as specifics such as age and gender of the mice, cohort size, and controls. Curr. Protoc. Mouse Biol. 1:17‐53. © 2011 by John Wiley & Sons, Inc.

Keywords: phenotyping; tissue collection; histology; necropsy; pathology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Necropsy for Histopathological Analysis
  • Basic Protocol 2: Fixation for Routine Histopathological Analyses with Formalin
  • Alternate Protocol 1: Fixation for Routine Histopathological Analyses with Bouin's Solution
  • Support Protocol 1: Trimming Organs and Defining Planes of Section
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Necropsy for Histopathological Analysis

  Materials
  • Mice (see Strategic Planning)
  • 10% (v/v) neutral buffered formalin (equivalent to 4% [w/v] formaldehyde; see recipe)
  • 70% ethanol (see recipe)
  • Plastic embedding cassettes (e.g., Fisher Scientific)
  • Large (≥1 liter) containers with lids (e.g., Fisher Scientific)

Basic Protocol 2: Fixation for Routine Histopathological Analyses with Formalin

  Materials
  • Mice (see Strategic Planning)
  • Bouin's fixative solution (e.g., VWR International LLC )
  • 70% ethanol (see recipe)
  • Plastic embedding cassettes (e.g., Fisher Scientific)
  • Large containers with lids (e.g., Fisher Scientific)
  • Rocking platform
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Figures

  •   FigureFigure 1. Proposed flow scheme for standardized, systematic post‐mortem analysis of a cohort of 24 mice. Step 1: Each age group is subdivided into two subgroups on the day of necropsy. Mice from subgroup A are subjected to systematic macroscopic and microscopic analyses. What remains of the paraffin blocks after sectioning is archived in a paraffin tissue bank. Mice from subgroup B are subjected to systematic necropsy and to a targeted histological evaluation. At this point, the subgroup B organs destined for paraffin embedding are the ones carrying a macroscopically visible defect in at least one of the mutant or treated mice. The remaining organs and the carcasses of the mice belonging to subgroup B are kept in buffered formalin until the completion of the histological screen (transient collection of mouse cadavers). Step 2:. A preliminary histopathological report will be available in 2 weeks after necropsy. Step 3: Depending on the histological findings, a second round of targeted histological analysis of subgroup B mice may be undertaken with the objective of increasing the statistical significance of the preliminary observations. Organs of subgroup B analyzed by histology at this stage are the ones in which histological defects have been detected in the subgroup A mutant or treated mice. To this purpose, these organs are collected from the bank of mouse cadavers, processed, embedded, and then stained with hematoxylin and eosin.
  •   FigureFigure 2. Steps following necropsy and fixation in histopathological analyses of mouse tissues.
  •   FigureFigure 3. Identification of mice by tattooing digits and ears. This figure proposes a numbering code for the permanent identification of 2‐week‐old to adult mice using forelimb digits for units, hindlimb digits for tens, and ears for hundreds. Units are tattooed by ink injection of forelimb digits, starting the count with 1 on the outermost right (anatomically right digit V) and progressing towards the outermost left (left digit V). Number 5 is obtained by tattooing both digits III and IV. Note that forelimb digits I are much reduced in size and therefore impossible to tattoo. Tens are tattooed on hindlimb digits, starting the count with 10 on the outermost right (anatomically right digit V) and progressing towards the outermost left (anatomically left digit V). As to hundreds, numbers 200 and 400 are indicated by tattooing dots on the right and left ears, respectively. Dots on both right and left ears indicate 600 (i.e., 200 plus 400). Units, tens, and hundreds are then added.
  •   FigureFigure 4. Template for necropsy report.
  •   FigureFigure 5. (A) Mouse with shaved back. (B) Mouse pinned ventral‐side‐up to cork board.
  •   FigureFigure 6. (A) Midline incision with skin reflected. Dissection of (B) salivary glands and (C) male preputial gland.
  •   FigureFigure 7. Dissection of mammary gland. (A) Insertion of scissors between the skin and the mammary gland, (B) spreading scissors to dissect the mammary gland from the skin, and (C) sectioning remaining adherences.
  •   FigureFigure 8. Dissection of pancreas.
  •   FigureFigure 9. Fixation of intestine.
  •   FigureFigure 10. Dissection of liver.
  •   FigureFigure 11. Dissection of kidneys.
  •   FigureFigure 12. (A) Dissection of testis, (B) urinary bladder, (C) seminal vesicle, and (D) prostate.
  •   FigureFigure 13. Dissection of (A) paragenital fat in the female, (B) ovaries and oviduct, and (C,D) bladder, vagina, and uterus (C,D)
  •   FigureFigure 14. Dissected ovaries, oviduct, bladder, vagina, and uterus.
  •   FigureFigure 15. Dissection of (A) adrenal glands and (B) mesenteric lymph nodes.
  •   FigureFigure 16. Dissection of aorta.
  •   FigureFigure 17. Removal of neck muscles (A,B) and view of exposed thyroid and trachea (C).
  •   FigureFigure 18. (A,B) Injection of fixative through trachea for fixation of lungs. (C) Lateral view of trachea with esophagus and thyroid.
  •   FigureFigure 19. Dissection of gastrocnemius and soleus muscles.
  •   FigureFigure 20. Dissection of brown adipose tissue (BAT).
  •   FigureFigure 21. Dissection of knee joint.
  •   FigureFigure 22. Incision through occipital bone for dissection of brain.
  •   FigureFigure 23. Removal of brain. (A) Placement for insertion of scissors tips, (B,C) splitting of the skull along medial suture, and (D) removal of skull halves.
  •   FigureFigure 24. Brain reflected towards anterior, showing optic chiasma and hypophysis underneath.
  •   FigureFigure 25. (A) Placement of brain in brain matrix and (B) position of razor blades for cutting slices.
  •   FigureFigure 26. Dissection of tail skin. (A) Longitudinal cut and (B) removal of spine.
  •   FigureFigure 27. Applications and limits of routine buffered formalin fixation. In general, a solution of 10% neutral buffered formalin preserves tissues appropriately and provides equal or higher quality for tissue stained with hematoxylin‐eosin than formalin‐substitute fixatives, the testis presenting a notable exception. (A) Secretion granules in Paneth cells (arrows), located within the intestinal crypts, are clearly identifiable after formalin fixation but (B) are extracted and thus appear as empty vacuoles after immersion in an acetic acid–alcohol proprietary fixative. (C) The seminiferous epithelium of the testis (here stained with hematoxylin and eosin) appears much more “life‐like” after Bouin's fixation than (D) after formalin fixation. Scale bar in D: 20 µm (A‐D).

Videos

Literature Cited

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Internet Resources
  http://www.eumorphia.org/
  The EUMORPHIA Web site, provides information about understanding human disease through mouse genetics.
  http://www.pathbase.net/
  Pathbase, a database of histopathology photomicrographs and macroscopic images derived from mutant or genetically manipulated mice.
  http://ctrgenpath.net/static/atlas/mousehistology/
  Provides an electronic atlas for the anatomy and histology of the mouse.
  http://www.deltagen.com/target/histologyatlas/HistologyAtlas.html
  Provides an electronic atlas for the histology of the mouse.
  http://www.dundee.ac.uk/histology/welcome.html
  A workbook with a set of microscope slides that illustrate the basic tissues and organs of the body.
  http://www.histosearch.com/
  Histosearch is a search engine that searches over 20,000 Web pages from histology‐related sites.
  http://stainsfile.info/StainsFile/jindex.html
  StainsFile is a useful site with general information and resources for histology.
  http://swehsc.pharmacy.arizona.edu/exppath/resources/formaldehyde.html
  Information on formaldehyde fixatives at the Southwest Environmental Health Sciences Center (SWEHSC), University of Arizona College of Pharmacy.
  http://www.ccac.ca/
  The Canadian Council on Animal Care (CCAC) is the national organization responsible for setting and maintaining standards for the care and use of animals in science in Canada.
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