Protocol - Fixation and paraffin embedding of tissue samples
It can be used for fixing and wax embedding of fresh samples.
Category:
Histology
Last revision: Jan 12, 2016
Contact
Name: Giovanna L. Liguori
Address: Institute of Genetics and Biophysics Via Pietro Castellino, 111 Naples 80131 ITALY
Email: giovanna.liguori@igb.cnr.it
Figure legend:
Transverse section of adult mouse intestine
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Dissect the embryos (or other sample) in PBS 1X
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Wash (for a few minutes) in PBS 1X, doing more than one change if necessary
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Fix the embryos in 4% paraformaldehyde (PFA) in 1X PBS
|
4° C
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over night (minimum 16 hours)
|
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Wash embryos twice in saline solution (NaCl 0.83%)
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RT
|
30 minutes
|
The isotonic solution (NaCl 0.83%) allows to remove residual fixative and phosphate which would otherwise be precipitated in the subsequent passages in ethanol.
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|
Dehydrate once with saline solution: 70% ethanol (1:1 ratio)
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RT
|
30 minutes
|
|
|
Dehydrate twice in 70% ethanol
|
RT
|
30 minute
|
after the second treatment with ethanol embryos can be stored indefinitely at 4 ° C
|
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To proceed with the inclusion of the embryos, dehydrate them with increasing concentrations of 85%, 95% ethanol and twice in 100% ethanol
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RT
|
30 minutes each
|
It is possible to leave the samples in the second 100% ethanol over night at 4°C
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Replace the ethanol with toluene twice
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RT
|
30 minutes
|
|
|
Replace with a mixture toluene: paraffin (1:1 ratio)
|
60° C
|
30 minutes
|
60°C melting point of the paraffin wax. Calculating the time since the wax is dissolved in toluene and shake to make homogenous mixture.
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Replace with three washes of paraffin
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60° C
|
30 minutes
|
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Transfer the sample into a mould previously filled with paraffin and always kept at 60 ° C. Orient the sample with the tip of a needle heated over the flame, holding the mould on a surface of about 60 ° C.
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After solidification, the sample can be stored (even indefinitely) at 4°C
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Other informations
Treatment time depends on the size of the sample, the protocol is set for E 9.5 embryos and treatment time have to be adjusted according to the size.
Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
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