Protocol for Preparation of Skin Tissue and Rafts before Paraffin Processing

*This protocol is for skin tissue and rafts. Please consult if using other tissues or if novel treatments are performed.

**Treatments (i.e. media, chemicals, etc…) affect how the samples should be fixed and handled. Please explain how the tissue was treated prior to dropping off samples. Helpful information includes:

o  Harvesting location of the tissues and/or cells and orientation of the tissue

o  How the tissue was harvested, including if perfusions were performed

o  How the raft was made (i.e. collagen or fibrin base)

o  If the tissue is a graft or wound (IMPORTANT)

o  Chemicals/drugs/media/etc… that the tissue was treated with

o  The type of fixative used, including when the fixative was added, duration of the fixative, and if the fixative has been changed during the course of fixation or if another chemical was added during fixation (i.e. formalin to ethanol to formalin)

o  How the tissue will be handled after sectioning (i.e. routine and special stains, IHC/ICC, IF, ISH, molecular procedures needing DNA/RNA/protein retrieval)

o  What to look for under the microscope (i.e. if a certain tissue layer needs to be present)

Protocol (for Paraffin):

  1. Though this step may vary depending on the experiment, samples should be fixed as soon as possible in order to preserve the best morphology.
  2. If using formalin, 10% Neutral Buffered Formalin is strongly recommended. If the formalin is unbuffered, too acidic, contaminated, or expired, black deposits can form on the samples and may interfere with section quality.
  3. The amount of formalin added must be 10 times the volume of the tissue.
  4. Some labs recommend changing the formalin after the first 30 minutes to 1 hour particularly if there is blood around the biopsy or sample.
  5. Fixation (and length of fixation) will interfere with some IHCs/IFs.
  6. If using paraformaldehyde, the paraformaldehyde must be made shortly before use. Storing paraformaldehyde significantly decreases the effectiveness of the fixation.
  7. Please consult if using paraformaldehyde, since the first step in the tissue processor is formalin.
  8. This step may also vary depending on the experiment: It is best to leave samples in formalin and do not transfer to 70% ethanol.
  9. Please contact the SDRC in order to coordinate when the samples need to be processed to make sure that the samples will not be left in formalin for too long.
  10. When dropping samples off in room 4-750, please leave samples under the fume hood and label the container with the solution, whether the sample is a tissue or raft, and name.
  11. You or the SDRC can place rafts and tissues into cassettes. If you have not been trained, contact the SDRC. Please specify the orientation of the tissue, including how the tissue was oriented on the animal and which side needs to be face down (cut first) on the cassette.
  12. If you would like the SDRC to cut (gross) the tissue, please meet with the SDRC first to show how you would like the tissue to be cut and oriented.
  13. If you are cutting the sample, a straight edge must be made. If you are unsure if your edge is straight enough or are having trouble, contact the SDRC. An edge that is not straight will decrease the quality of the section and may alter the morphology.
  14. Procedure for placing samples into cassettes:
  15. Fill a container/jar with a secure lid (the plastic containers from VWR work great, the SDRC will have some as well) with enough fixative (or solution in not storing in fixative) to cover all cassette samples.
  16. Use and label a regular/slit cassette with a pencil in dark writing. Do not use a biopsy or mega cassette. If you are using your own cassettes, check with the SDRC first to make sure your cassette is compatible with the tissue processor and microtome.
  17. Include your name/initials, the title/number of the sample, experiment number/code, and date (optional).
  18. Two biopsy sponges will be used. Pre-soak the sponges in formalin (unless using a different solution, soak in the solution that the cassettes will be placed in) prior to placing in the cassettes. *The sponges may need to be trimmed in order to properly fit into the cassette. Sponges and sample must be flat.
  19. Trim your sample into a rectangle that will fit into the cassette. You may leave the raft as-is if the raft is small enough to fit into the cassette. Use a sharp blade and press down until you have cut through the sample. Taping the top of the blade with forceps can help. Do not drag or wriggle the blade as this will create a jagged edge. Sometimes, using a blade and scalpel will be needed.
  20. Please contact the SDRC for training to get the raft off of the grid. The rafts stick to the grid and improper handling can rip the raft. Contact the SDRC if you have not cut a raft before.
  21. Place one sponge in the cassette. Place your sample in the cassette. Place another sponge on top of your sample and close the cassette lid gently. Place the cassette into the container/jar. Label the jar with the date, your name, and the solution.
  22. Place excess solutions in the proper waste containers under the fume hood.
  23. *Do not use the bio-wraps unless otherwise noted.

Solutions used during tissue processing

  1. 10% buffered neutral formalin
  2. Alcohol dehydrations from 70% to 100%
  3. Sub-X clearing agent (a xylene substitute)
  4. Paraffin (Paraplast Extra)