Difference between revisions of "Paraffin Embedding of Tissue Samples"

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==SOP==
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*[[SOP - Carcinogens]]
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*[[SOP - Flammables and Combustibles]]
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*[[SOP - Formalin and Paraformaldehyde]]
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*[[SOP- Irritants]]
  
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== Protocol ==
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== Paraffin embedding protocol ==
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#After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
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#Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
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#After fixation, begin dehydrating the tissue in 70% Ethanol
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#Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required. At this step samples can also be taken to the [https://www.rogelcancercenter.org/research/shared-resources-and-cores/tissue-and-molecular-pathology Rogel Cancer Center's Tissue and Molecular Pathology Core] for the remaining steps
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#On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
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##Note that the subsequent dehydration steps involve many long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
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##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
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#Turn on wax pots.
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#After 100% Ethanol washes, Wash 3 times in Xylenes
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#Move cassettes into 75% ethanol for 30 minutes.
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#Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
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#Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
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#Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
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#Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
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#Take your sample and orient it on top of the base wax as desired
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##Some protocols suggest that the first 100% ethanol wash can be left over night if time is an issue. However, it must be noted that extending the dehydration process can result in alterations to the morphology of your tissue.
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#Place pathology cassette on top of the mold and fill with wax covering the mold
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##During these incubation steps, prepare beakers with 58-60 deg C paraffin (the melting point of paraffin is 58 C; many embedding procedures recommend that the paraffin to be 2 C above the melting point for best results).
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#Place mold with sample on "Cold Side" of embedding station  
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#Move cassettes into Citrosolv for 30-60 minutes. Repeat this step a second time, using fresh Citrosolv.
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##Wax will solidify in 10-15 minutes
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#Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time.
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#Remove wax embedded sample from the mold (should just slip out)  
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##Some protocols suggest that the second paraffin incubation step can be extended over night. However, this may increase the risk of the tissue cracking during sectioning.
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#Store at room temperature until prepped to section
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##During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
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#Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
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#Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
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#Place pathology cassette on top of the mold and completely fill the mold.
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#Place mold with sample on "Cold Side" of embedding station .
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##The paraffin will solidify in 10-15 minutes.
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#Remove embedded sample from the mold (should just slip out).
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#Store at room temperature until ready to section.
  
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==Wash Solution Table==
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Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
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|-
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1. Collect tissue
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!style="border-style: solid; border-width: 0 1px 1px 0"| Solution
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a. Immediately place in labeled cassette in 10% buffered formalin
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!style="border-style: solid; border-width: 0 0 1px 0"| Time
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b. Place in labeled cassette on dry ice prior to storage at -80C
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| 70% ethanol
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2. Fix in 10% buffered formalin
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|style="border-style: solid; border-width: 0"| Overnight/24 hours
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a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| 75% ethanol
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3. Tissue processing
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|style="border-style: solid; border-width: 0"| 30 min
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a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage
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|-
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b. 95% EtOH (30 min)
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|style="border-style: solid; border-width: 0 1px 0 0"| 95% ethanol
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c. 100% EtOH (30-60 min)
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|style="border-style: solid; border-width: 0"| 75 min
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d. Xylene (30 min under a fume hood)
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|-
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e. Xylene (30 min under a fume hood)
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|style="border-style: solid; border-width: 0 1px 0 0"| 95% ethanol
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f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
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|style="border-style: solid; border-width: 0"| 75 min
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g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| 100% ethanol
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*NOTE: Steps 3a to 3e can be done in bulk using plastic histology containers placed on a shaker. In steps 3f to 3g transfer the tissue to a 1.5 ml labeled eppendorf tube and add melted Paraffin → place in a 56 oC heated water bath and floating tube rack.
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|style="border-style: solid; border-width: 0"| 60 min
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|-
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4. Embedding
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|style="border-style: solid; border-width: 0 1px 0 0"| 100% ethanol
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a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax.
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|style="border-style: solid; border-width: 0"| 60 min
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b. Pour a small amount (1/3 of the volume) of heated wax into a pre-heated stainless steel cassette and arrange tissue pressing it flat against the bottom with a pre-heated instrument.
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|-
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c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh.
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|style="border-style: solid; border-width: 0 1px 0 0"| 100% ethanol
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d. Chill until paraffin is completely solidified and then remove stainless steel cassette.
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|style="border-style: solid; border-width: 0"| 60 min
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Sectioning
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|style="border-style: solid; border-width: 0 1px 0 0"| Citrosolv
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1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
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|style="border-style: solid; border-width: 0"| 30 min
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2) Pretreatment of paraffin tissue sections with either of the following
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|-
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a. Leave slides at room temperature for 60 minutes; or
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|style="border-style: solid; border-width: 0 1px 0 0"| Citrosolv
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b. Heat in dry oven at 55°C for 20 minutes
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|style="border-style: solid; border-width: 0"| 30 min
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Note 1: for adipose, leaving at room temperature prevents disruption of the tissue
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|-
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Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules.
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|style="border-style: solid; border-width: 0 1px 0 0"| 60 degree paraffin
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3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
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|style="border-style: solid; border-width: 0"| 60 min
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4) Immerse slide in 100% ethanol for 2 minutes.
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|-
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5) Immerse slide in 95% ethanol for 1 minute.
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|style="border-style: solid; border-width: 0 1px 0 0"| 60 degree paraffin
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6) Immerse slide in 70% ethanol for 1 minute.
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|style="border-style: solid; border-width: 0"| 60 min
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7) Immerse slide in 50% ethanol for 1 minute.
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|-
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8) Immerse slide in 1X PBS for 2 minutes.
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|style="border-style: solid; border-width: 0 1px 0 0"| 60 degree paraffin
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9) Air dry slides for 10-20 min
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|style="border-style: solid; border-width: 0"| 60 min or O/N
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|}
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Protocol Edited from: several online IHC protocols
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H&E Staining
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Hematoxylin staining
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Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver)
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Rinse in warm H2O (note ‘blueing’ of nuclei)
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Eosin staining
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Working solution:  
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• 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol)
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• 75 mL of 80% Ethanol
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• 0.5 mL Glacial Acetic Acid
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Stain sections for 10 min. in 0.25% Eosin solution
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Rinse slides in ddH2O
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Dehydrate slides in
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1) Immerse slide in 70% ethanol (4 dips)
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2) Immerse slide in 95% ethanol (2 dips).
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3) Immerse slide in 100% ethanol (2 dips).
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4) Immerse slide in xylene for (6 dips)
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Mounting
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Apply a drop of mounting medium over the section
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Place the cover slip over in an angle to avoid bubbles
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Store slides at room temperature for 2 h prior to analysis
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Latest revision as of 15:49, 5 December 2023

SOP

Paraffin embedding protocol

  1. Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
  2. Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required. At this step samples can also be taken to the Rogel Cancer Center's Tissue and Molecular Pathology Core for the remaining steps
    1. Note that the subsequent dehydration steps involve many long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
  3. Turn on wax pots.
  4. Move cassettes into 75% ethanol for 30 minutes.
  5. Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
  6. Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
    1. Some protocols suggest that the first 100% ethanol wash can be left over night if time is an issue. However, it must be noted that extending the dehydration process can result in alterations to the morphology of your tissue.
    2. During these incubation steps, prepare beakers with 58-60 deg C paraffin (the melting point of paraffin is 58 C; many embedding procedures recommend that the paraffin to be 2 C above the melting point for best results).
  7. Move cassettes into Citrosolv for 30-60 minutes. Repeat this step a second time, using fresh Citrosolv.
  8. Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time.
    1. Some protocols suggest that the second paraffin incubation step can be extended over night. However, this may increase the risk of the tissue cracking during sectioning.
    2. During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
  9. Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
  10. Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
  11. Place pathology cassette on top of the mold and completely fill the mold.
  12. Place mold with sample on "Cold Side" of embedding station .
    1. The paraffin will solidify in 10-15 minutes.
  13. Remove embedded sample from the mold (should just slip out).
  14. Store at room temperature until ready to section.

Wash Solution Table

Solution Time
70% ethanol Overnight/24 hours
75% ethanol 30 min
95% ethanol 75 min
95% ethanol 75 min
100% ethanol 60 min
100% ethanol 60 min
100% ethanol 60 min
Citrosolv 30 min
Citrosolv 30 min
60 degree paraffin 60 min
60 degree paraffin 60 min
60 degree paraffin 60 min or O/N
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php?title=Paraffin_Embedding_of_Tissue_Samples&oldid=2685"