alligator snapping turtle
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Paraffin EmbeddingParaffin Embedding
Protocol of Turtles tailProtocol of Turtles tailSampleSample
AlaadinAlaadin AlayoubiAlayoubi
2828thth Sep 2010Sep 2010
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The Alligator Snapping TurtleThe Alligator Snapping Turtle
((MacrochelysMacrochelys temminckiitemminckii)) one of the largest freshwater turtles in theone of the largest freshwater turtles in the
worldworld
The largest freshwater turtle in North AmericaThe largest freshwater turtle in North America
(They weigh between 155 and 175 pounds)(They weigh between 155 and 175 pounds)
It looks very primitive and has been called theIt looks very primitive and has been called the
dinosaur of the turtle worlddinosaur of the turtle world
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TissueTissue of interestof interest
Focusing will be on the tail of the turtleFocusing will be on the tail of the turtle
the tail has circular shape at the base andthe tail has circular shape at the base and
then become flat towards the tipthen become flat towards the tip
Our goal is to figure out any difference in theOur goal is to figure out any difference in the
histology of the two parts of the tailhistology of the two parts of the tail
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SpecimenSpecimen
Tail is an extension of the spinal cordTail is an extension of the spinal cord
Contains several tissues: Bones , muscleContains several tissues: Bones , muscle
tissues and connective tissuestissues and connective tissues
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ProcessingProcessing protocolprotocol
FixationFixation
DecalcificationDecalcification
DehydrationDehydration
Paraffin embeddingParaffin embedding
SectioningSectioning
StainingStaining
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FixationFixation
It is already fixed in 10%It is already fixed in 10%buffered formalinbuffered formalin
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All fixed specimens are washed in slowlyAll fixed specimens are washed in slowlyrunning tap water for a minimum of 30running tap water for a minimum of 30minutes.minutes.
Solutions :Solutions :
1.1. Hydrochloric Acid/Formic Acid WorkingHydrochloric Acid/Formic Acid WorkingSolution :Combine equal parts of the 8%Solution :Combine equal parts of the 8%hydrochloric acid solution and the 8%hydrochloric acid solution and the 8%
formic acid solution before use.formic acid solution before use.2.2. Ammonia Solution:Ammonia Solution:
DecalcificationDecalcification
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Procedure:Procedure:
Change to fresh solution each day untilChange to fresh solution each day until
decalcification is complete. It may take 24decalcification is complete. It may take 24
hours up to days or months depending on sizehours up to days or months depending on size
of the specimens.of the specimens.
testing procedures determine the end pointtesting procedures determine the end point
(Decalcification for rabbit spine using this(Decalcification for rabbit spine using this
protocol for overnight about 20 hours and itprotocol for overnight about 20 hours and itshowed very good result)showed very good result)
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Once the decalcification is complete, rinseOnce the decalcification is complete, rinsespecimens in water briefly and transfer tospecimens in water briefly and transfer toammonia solution to neutralize acids left inammonia solution to neutralize acids left inspecimens for 30 minutes.specimens for 30 minutes.
Wash specimens in running tap waterWash specimens in running tap waterthoroughly up to 24 hours.thoroughly up to 24 hours.
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EndEnd--Point of Decalcification:Point of Decalcification:
XX--ray (the most accurate way)ray (the most accurate way)
Chemical testing (accurate):Chemical testing (accurate):
Ammonium Hydroxide/Ammonium OxalateAmmonium Hydroxide/Ammonium Oxalate
Working SolutionWorking Solution
Physical testing (less accurate): bending thePhysical testing (less accurate): bending thespecimenspecimen
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DehydrationDehydration
70% ethanol, 2 changes, 1h each70% ethanol, 2 changes, 1h each
80% ethanol, 2 changes, 1h each80% ethanol, 2 changes, 1h each
95% ethanol, 2 changes, 1h each95% ethanol, 2 changes, 1h each
100% ethanol, 3 changes, 1h each100% ethanol, 3 changes, 1h each
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Infiltration & embeddingInfiltration & embedding
Xylene: 3 changes, 1h eachXylene: 3 changes, 1h each
paraffin wax (56paraffin wax (56--58C), 2 changes, 1.5h each58C), 2 changes, 1.5h each
embed tissues into paraffin blocksembed tissues into paraffin blocks
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SectioningSectioning
Section thickness 5 mSection thickness 5 m --10 m10 m
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RehydrationRehydration
xylene (twice) 5 minxylene (twice) 5 min
100%100% EtOHEtOH(Twice) 5min(Twice) 5min
95%95% EtOHEtOH(Twice) 4min(Twice) 4min
80%80% EtOHEtOH 4min4min
Tap water 4 minTap water 4 min
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StainingStaining
hematoxylin and eosinhematoxylin and eosin
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ReferencesReferences VerdeniusVerdenius HHW and Alma L (1958) A quantitative study of decalcificationHHW and Alma L (1958) A quantitative study of decalcification
methods in histology Jmethods in histology J ClinClin PatholPathol. 1958 May; 11(3): 229. 1958 May; 11(3): 229236.236.
Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008a. ParaffinFischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008a. Paraffin
embedding tissue samples for sectioning.embedding tissue samples for sectioning. CSH ProtocolsCSH Protocols (this issue)(this issue) doidoi::
10.1101/pdb.prot498910.1101/pdb.prot4989
Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008b.CuttingFischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008b.Cutting
sections of paraffin embedded tissues.sections of paraffin embedded tissues. CSH ProtocolsCSH Protocols (this issue)(this issue) doidoi::
10.1101/pdb.prot4987.10.1101/pdb.prot4987.
Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008c.Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008c.
Decalcifying tissues for paraffin embedding.Decalcifying tissues for paraffin embedding. CSH ProtocolsCSH Protocols (this issue)(this issue)doidoi: 10.1101/pdb.prot4990.: 10.1101/pdb.prot4990.
PresnellPresnell J.K andJ.K and SchreibmanSchreibman M.P.M.P. 1997.Humasons1997.Humasons AmimalAmimal TissueTissue
TechniquesTechniques, Fifth ed. The Johns Hopkins University Press, Baltimore,, Fifth ed. The Johns Hopkins University Press, Baltimore,
LondonLondon