Fixation and Processing of mouse tissue

GENERAL:

  1. No ONE fixative works for all antigens and all antibodies. Conditions must be optimized for each antigen-antibody combination.
  2. Fixation should be rapid but thorough.
    1. The optimal tissue size is no thicker than 1-2mm. If a tissue is thicker, the investigator should fix for one hour, and then cut 1mm thick sections (“bread-loaf”) so that all surfaces are exposed equally.
    2. The Volume of fixative should be at least 10x the volume of the tissue. If the tissue is “bloody”, remove the bloody fixative after an hour and add fresh fixative.
    3. A tissue can be placed in one fix and then portions placed in a second fixative for “mordanting”. (Example: fix in paraformaldehyde for ISH. Place a paraformaldehyde sample in Bouin’s Fix for IHC.)
  3. Once the tissue is fixed, it should be held in 70% alcohol and processed promptly.
  4. Paraformaldehyde is the best fixative for ISH. Bouin’s Fixative is the best all-around fixative for IHC. (Do not use the Bouin’s substitute without Picric Acid. The Picric Acid is required for best results.)

PROTOCOL:

  1. Remove tissue as rapidly as possible. Place in 10 volumes of fixative. Cold fixative is better than warm or room temperature fixative.
  2. After one hour, inspect sample. If the fixative is cloudy, replace with fresh fixative. If the sample is thick, remove sample, place on a cutting board, cut 1-2mm slices to expose all surfaces and return to 10 volumes of fixative. (note: the short fixation will firm up hard-to-slice tissues such as lymph nodes, spleen and thymus, making them easier to slice).
  3. Fix for six to 12 hours of over night BUT no longer. Remove and place in 70% ETOH until shipping or processing.

SAMPLING:

  1. If the tissue is of interest, make sure that at least 50% of the sample is composed of normal adjacent tissue.
  2. In the case of the mammary gland, the gland should be spread on a slide so that the fat does not inhibit fixation. After an hour, the flattened mammary gland can be removed from the slide and placed in more fixative.
  3. If one organ is diseased, make sure that the contralateral organ is sampled (if the organs are bilateral).
  4. The best interpretation is provided when age matched control organs are provided.

INFORMATION:

  1. Make sure that the sample is submitted with the appropriate request forms that includes demographic and experimental data about the animal.