I am trying to do some histology studies for lungs that were extracted from mice. These lungs have metastases and I have followed the standard protocol used here for the dehydration, fixation and embedding. When cutting the sample using the microtome the paraffin was being correctly sliced, however we would get a hole on the area of the tissue. The tissue was not being sliced together with the paraffin at all. Does anyone have any idea why this was happening? Any help would be really appreciated.
The protocol used is shown here:
- After extracting the lungs and the tumors from the animals, place them in a 10 % formalin solution (or 4% formaldehyde solution, diluted with PBS).
- Store the tubes in the refrigerator overnight.
- Replace the solution with 70 % ethanol. Store in the refrigerator. The specimens can be stored for several weeks.
- In order to start the paraffin embedding place the samples in a beaker filled with 80 % ethanol for 30 minutes. The samples must be thin enough in order to fit the mold.
- Transfer the samples into a 95 % ethanol solution and let incubate for 1h.
- Replace the solution with a 100 % ethanol solution and incubate for 1h30. The beaker should be covered with parafilm in order to avoid any evaporation.
- Incubate for 1h with Clear-Rite 3 (Richard-Allan Scientific) or xylene.
- Place the samples in a beaker filled with paraffin. This beaker must be placed in a 60 ºC oven prior to adding the samples in order to assure that the paraffin is already melted.
- Incubate for 1h30.
- After incubation, pour a little bit of the paraffin solution into the bottom of the mold allowing to obtain a thin layer (approximately 2-3 mm thick).
- Without waiting more than 30 seconds, place the sample in the mold paying particular attention to the orientation of the tissue. Place the orange lid on top of the mold and quickly fill it with the melted paraffin. Make sure there are no bubbles and that there is enough paraffin above the lid in order to leave approximately a layer of about 3-4 mm on top. After pouring the paraffin tap the mold against a surface a couple of times in order to make sure that the solution is uniformly distributed.
- Place the molds on the cooling surface for 30 minutes.
- Store the molds in the -20 ºC freezer overnight.
- Remove the molds from the freezer.
- Install the blade (which is stored in the wood box located in drawer below the desk where the microtome is) on the microtome.
- Make sure the orientation is like 45 º for the blade. That can be adjusted in order to optimize the slicing of the specimens.
- Adjust the cutting thickness to 7-8 microns.