Paraffin Embedding

Protocol of Turtle’s tail

Sample

Alaadin Alayoubi
28th Sep 2010
 The Alligator Snapping Turtle
(Macrochelys temminckii)
one of the largest freshwater turtles in the world

The largest freshwater turtle in North America

(They weigh between 155 and 175 pounds)

It looks very primitive and has been called the

dinosaur of the turtle world

 Tissue of interest

 Focusing will be on the tail of the turtle

 the tail has circular shape at the base and

then become flat towards the tip

 Our goal is to figure out any difference in the

histology of the two parts of the tail

 Specimen

 Tail is an extension of the spinal cord

 Contains several tissues: Bones , muscle

tissues and connective tissues

 Processing protocol
 Fixation
 Decalcification
 Dehydration
 Paraffin embedding
 Sectioning
 Staining
 Fixation

It is already fixed in 10%

buffered formalin
 Decalcification
  All fixed specimens are washed in slowly
running tap water for a minimum of 30
minutes.

 Solutions :       
1. Hydrochloric Acid/Formic Acid Working
Solution :Combine equal parts of the 8%
hydrochloric acid solution and the 8% formic
acid solution before use.
2. Ammonia Solution:

      
Procedure:

 Change to fresh solution each day until

decalcification is complete. It may take 24
hours up to days or months depending on size
of the specimens.

 testing procedures determine the end point

(Decalcification for rabbit spine using this
protocol for overnight about 20 hours and it
showed very good result)
 
 Once the decalcification is complete, rinse
specimens in water briefly and transfer to
ammonia solution to neutralize acids left in
specimens for 30 minutes.

 Wash specimens in running tap water

thoroughly up to 24 hours.
End-Point of Decalcification:

 X-ray (the most accurate way)

 Chemical testing (accurate):

Ammonium Hydroxide/Ammonium Oxalate
Working Solution

 Physical testing (less accurate): bending the

specimen
 Dehydration

70% ethanol, 2 changes, 1h each

80% ethanol, 2 changes, 1h each

95% ethanol, 2 changes, 1h each

100% ethanol, 3 changes, 1h each

 Infiltration & embedding

 Xylene: 3 changes, 1h each

 paraffin wax (56-58ºC), 2 changes, 1.5h each

 embed tissues into paraffin blocks

 Sectioning

Section thickness 5 µm -10 µm

 Rehydration

 xylene (twice) 5 min

 100% EtOH(Twice) 5min
 95% EtOH(Twice) 4min
 80% EtOH 4min
 Tap water 4 min
 Staining

hematoxylin and eosin

 References
 
Verdenius HHW and Alma L (1958) A quantitative study of decalcification
methods in histology J Clin Pathol. 1958 May; 11(3): 229–236.
    
Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008a. Paraffin

embedding tissue samples for sectioning. CSH Protocols (this issue) doi:
10.1101/pdb.prot4989

Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008b.Cutting

sections of paraffin embedded tissues. CSH Protocols (this issue) doi:
10.1101/pdb.prot4987.

Fischer, A.H., Jacobson, K.A., Rose, J., and Zeller, R. 2008c. Decalcifying
tissues for paraffin embedding. CSH Protocols (this issue) doi:
10.1101/pdb.prot4990.

PresnellJ.K and Schreibman M.P. 1997.Humason’s Amimal Tissue

Techniques, Fifth ed. The Johns Hopkins University Press, Baltimore,
London