Histology Lab YCCMD Tissue Processing Options

Traditional MMA – 4 weeks

Harvest bone immediately from euthanized animal, scrape clean of all soft tissue and immerse immediately in 70% ethanol at a ratio of 20X volume of fixative vs. bone.  Place in refrigerator at 4˚C (do not freeze) for up to one month.

Advantages:

  • Ideal for bones, better bone cell (osteoblasts, osteoclasts) detail than rapid MMA.
  • Can remove MMA completely prior to staining.

Disadvantages:

  • Takes longer.
  • Does not preserve enzyme/antigen activity as well.

Rapid MMA – 2 weeks

Harvest bone immediately from euthanized animal, scrape clean of all soft tissue and immerse immediately in 70% ethanol at a ratio of 20X volume of fixative vs. bone.  Place in refrigerator (do not freeze) for up to one month.

Advantages:

  • Speed, preserves alk/acid phosphatase activity and some antigenic activity.
  • Can remove MMA completely prior to staining.
Disadvantages:
  • For optimal section quality for photographs use traditional MMA method.

Please Note:

  • This method can only be used for mouse bones!
  • Bone (cleaned of soft tissue) may be fixed in 10% Neutral buffered formalin (20X volume of fixative relative to tissue size) for up to 48 hours at room temperature, washed in running water for at least 4 hours, then transferred to 70% ethanol then embedded in plastic via the Traditional MMA method or the Rapid MMA method. This method of fixation is optimal for hematopoietic cell detail. However, formalin decreases the quality of enzyme staining and bone cell detail.

Paraffin

Put fresh tissue into 10% buffered formalin (20X volume of fixative to tissue).  Soft tissue should be sliced to the thickness of a nickel (coin) prior to immersion in fixative for optimal penetration of fixative and should not be left in formalin for more than 24 hours to prevent overfixation and hardening of tissue.

Advantages:

  • Ideal for soft tissue, less cost, quicker turnaround time, better for immunostaining methods.

Disadvantages:

  • Must decal bone for this procedures, decalcification will cause the loss of any FL bone labels. Paraffin will not support any hard tissue (bone) or implant.

GMA (glycolmethacrylate)

Place fresh tissue into either 10% neutral buffered formalin (NBF) at room temperature or 70% ethanol at 4˚C (20X volume of fixative to tissue).  Tissue should not be left in NBF for more than 24 hours to prevent overfixation and hardening of tissue.

Advantages:

  • Speed, preserves enzyme/antigen activity, works well for soft tissue with non metal implants.

Disadvantages:

  • GMA is water soluble, less reliable plastic: can only be used for small specimens.
  • GMA does not support fully mineralized bone tissue well, MMA is optimal for this.
  • GMA cannot be removed prior to staining but can be stained through.
Please Note:
  • For any specimens to be processed for histological purposes– DO NOT FREEZE!
  • Specimens with fluorescent labels (calcein) should be kept in the dark and should NOT be decalcified since this would remove the FL label.