Preparing Samples for Histological Analysis

Samples for paraffin embedding:

Make sure that the volume of formalin used for fixing the tissue is 20x the volume of the sample. Fix your samples in 10% neutral buffered formalin (NBF) or PFA for 24 hours at room temperature.  Fixing the tissue in the refrigerator will slow down and inadequately fix the tissue. Following fixation, replace the formalin with 70% EtOH and keep the sample at room temperature.  

Back skin (E17.5-adult mice): Within the same experiment, skin samples should be harvested from the same area of the body. Often, the back of the mouse is chosen for this purpose. If you are using back skin, take the biopsy from the middle of the back (on top of the spine) midway between the head and tail. Using a scissor or scalpel, trim the biopsy into a rectangle, such that the long axis of the rectangle corresponds to the A-P axis of the mouse. Unless otherwise indicated, the biopsy will be sectioned along the long axis of the rectangle. If sectioned this way, your sections will provide a good view of the interfollicular epidermis as well as the hair follicles. If you prefer sectioning along a different axis, please indicate this clearly on the requisition form. Place your rectangular piece of skin flat on a piece of Whatman filter paper or unlined index card (dermis side down). This will ensure that the skin sample will remain flat while it is fixing. Then place the paper with the skin between 2 blue biopsy pads and place in a labeled (with pencil) plastic tissue biopsy cassette.  Drop cassette(s) in a container of 10% neutral buffered formalin for a minimum of 24 hours.  Make sure to use 20x the amount of formalin per the amount of tissue to be fixed.

Embryonic skin (up to E16.5): For embryos up to E16.5, fix the entire embryo rather than attempting to remove the skin. Carefully dissect the embryo free from extraembryonic tissues, place it in 10% NBF or Bouin’s fixative, and proceed as above. Unless otherwise indicated, embryos will be sectioned from the midline along the sagittal axis. 

Ear skin: Remove the entire ear of an adult mouse and trim the biopsy into a rectangle, such that the long axis of the rectangle is perpendicular to the head of the mouse. This procedure using blue biopsy pads should also be used for mouse ears, to prevent the ear from curling up during fixation.

Tumors: After dissecting out the tumor, trim the tumor to prepare a biopsy with at least one straight edge. The straight edge will be the cutting edge of the sample. If possible, the cutting edge should be perpendicular to the skin of the mouse, such that your section will contain the most superficial as well as the most basal part of the tumor. 

Samples for OCT embedding:

If you have never embedded skin in OCT, please contact the core for help with your first sample. As a general guideline, prepare the skin sample as above (i.e. rectangular pieces of back skin, etc). Since the sample will be sectioned from the bottom of the cryomold, your sample should be placed in the cryomold accordingly. For example, rectangular pieces of back skin must be placed on the bottom of the cryomold on the edge of the long side. Slowly fill the cryomold with OCT and place the sample in the middle of the cryomold. Once you have positioned the biopsy in OCT, make sure to remove any bubbles that may surround your tissue, as they can cause problems with cutting. You can remove the bubbles with a thin needle. Place the cryomold + OCT + biopsy on dry ice to allow the OCT to freeze (it will turn white) or flash freeze in liquid nitrogen. Once the OCT is frozen, store your samples at -80°C. Embryos must be processed through a sucrose gradient prior to embedding in OCT.