Histology: preparation of samples for FFPE or frozen sectioning

Loiselle Lab Histology Protocol: preparation of samples for FFPE or frozen sectioning

FFPE (formalin-fixed paraffin embedded) samples

** Unless your experiment requires frozen sectioning (certain stains, endogenous fluorescence), FFPE should be the default: cheaper, better morphology, & easier to do co-immunofluorescence

1. Prepare sample cassettes – label with histology ID, brief description, as well as R/L hindpaw (make sure to use histology marking pen)
   • Log samples into the histology logbook, plus any additional details (tamoxifen tx etc)
2. Dissection procedure:
   • Clear as much skin as possible from the top of the paw using surgical scissors
   • Cut off the last bit of each toe
   • Sever hind paw from leg a few cm above the ankle with bone cutters
   • Place in labeled cassette, and immediately put in 10% NBF
3. Fix in 10% NBF for 72h at RT with rocking
4. Rinse 1x briefly with 1X PBS
5. Cover with PBS and wash 2x on rocker for 10’
6. Briefly rinse with dH2O, then cover with dH2O (if soft tissue, leave in 70% EtOH)
7. Fill out histology work order form, and deliver to core:
   • Samples will need a 2wk decal in Webb-Jee 14% EDTA, this can be done by us or the core (since they cannot always process the same day after decal it’s usually better to give samples to them at least a few days before decal is finished)
8. After the core processes the samples for paraffin, they’ll return them for us to embed

Frozen samples

1. Same preparation/dissection procedure as for FFPE
2. Fix in 10% NBF for 24h at 4C with shaking
3. Rinse with PBS 2x for 10’
4. Decal in Webb-Jee 14% EDTA 4 days at 4C with shaking
5. Rinse with PBS 2x for 10’
6. Incubate in 30% sucrose in PBS at 4C O/N with shaking 
7. At this point, can freeze samples at -80C in 30% sucrose in 1.5mL eppendorf tubes, or continue to embedding:
   • Acquire dry ice from the biological supply center, both flat pieces as well as smaller bits
   • Fill metal canister with methylbutane to ~3-4 inches, put in ice bucket and surround with dry ice.  Allow to chill ~30min
   • Incubate samples in cryomatrix for a few minutes to equilibrate
   • Transfer sample to labeled plastic mold on top of a ‘dot’ of cyromatrix
     - For hind paws, make sure medial side is DOWN (check histology logbook to verify R or L paw)
   • Place mold with sample on dry ice, and hold until hindpaw is frozen in place
   • Add more cryomatrix to completely cover sample
   • Lower into methylbutane to snap freeze, wrap each individually in foil, and then transfer to -20C until ready for sectioning