Alligator Snapping Turtle

8/8/2019 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





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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

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Alligator Snapping Turtle