Paraffin Embedding of Tissue Samples

1. After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
2. After fixation, begin dehydrating the tissue in 70% Ethanol
3. On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
   1. While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
4. After 100% Ethanol washes, Wash 3 times in Xylenes
5. Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
6. Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
7. Take your sample and orient it on top of the base wax as desired
8. Place pathology cassette on top of the mold and fill with wax covering the mold
9. Place mold with sample on "Cold Side" of embedding station
   1. Wax will solidify in 10-15 minutes
10. Remove wax embedded sample from the mold (should just slip out)
11. Store at room temperature until prepped to section

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.
   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. Move cassettes into 75% ethanol for 30 minutes.
4. Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
5. 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).
6. Move cassettes into xylenes for 30-60 minutes. Repeat this step a second time, using fresh xylenes.
7. 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).
8. Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
9. Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
10. Place pathology cassette on top of the mold and completely fill the mold.
11. Place mold with sample on "Cold Side" of embedding station .
    1. The paraffin will solidify in 10-15 minutes.
12. Remove embedded sample from the mold (should just slip out).
13. Store at room temperature until ready to section.

Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab

1. Collect tissue a. Immediately place in labeled cassette in 10% buffered formalin b. Place in labeled cassette on dry ice prior to storage at -80C

2. Fix in 10% buffered formalin a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin

3. Tissue processing a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage b. 95% EtOH (30 min) c. 100% EtOH (30-60 min) d. Xylene (30 min under a fume hood) e. Xylene (30 min under a fume hood) f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra) g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)

• 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.

4. Embedding a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax. 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. c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh. d. Chill until paraffin is completely solidified and then remove stainless steel cassette.

Sectioning 1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC). 2) Pretreatment of paraffin tissue sections with either of the following a. Leave slides at room temperature for 60 minutes; or b. Heat in dry oven at 55°C for 20 minutes Note 1: for adipose, leaving at room temperature prevents disruption of the tissue Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules. 3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver). 4) Immerse slide in 100% ethanol for 2 minutes. 5) Immerse slide in 95% ethanol for 1 minute. 6) Immerse slide in 70% ethanol for 1 minute. 7) Immerse slide in 50% ethanol for 1 minute. 8) Immerse slide in 1X PBS for 2 minutes. 9) Air dry slides for 10-20 min

Protocol Edited from: several online IHC protocols

H&E Staining

Hematoxylin staining Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver) Rinse in warm H2O (note ‘blueing’ of nuclei)

Eosin staining Working solution: • 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol) • 75 mL of 80% Ethanol • 0.5 mL Glacial Acetic Acid

Stain sections for 10 min. in 0.25% Eosin solution Rinse slides in ddH2O

Dehydrate slides in 1) Immerse slide in 70% ethanol (4 dips) 2) Immerse slide in 95% ethanol (2 dips). 3) Immerse slide in 100% ethanol (2 dips). 4) Immerse slide in xylene for (6 dips)

Mounting Apply a drop of mounting medium over the section Place the cover slip over in an angle to avoid bubbles Store slides at room temperature for 2 h prior to analysis