- First, place a tissue slice on a filter paper soaked with PBS to prevent cell shrinkage or swelling by balancing osmolarity. Transfer the tissue to a histo cassette. Close and place the cassette in neutral buffered formalin, NBF solution. NBF contains formaldehyde that binds to amino groups of amino acids and prevents formic acid formation that pigments the tissue. Leave the cassette overnight at a low temperature, which prevents nucleic acid degradation and fixes the tissue. Dehydrate the tissue with multiple washes of alcohol.
Place it in liquid paraffin and press it down to the mold's bottom. Remove the weight and place the bottom of the histo cassette on the tissue. Pour liquid paraffin on the assembly. Allow the setup to solidify on a cold plate. Paraffin is insoluble in water, and after entering the dehydrated tissue it hardens and helps in sectioning. Transfer the tissue block on a microtome. Cut and collect thin sections of the tissue on a slide. If the section is too thick, oxygen and other nutrients cannot diffuse across the slice creating a false necrosis gradient. If the section is too thin, then it tends to curl. The protocol video examines biomarker expression and cell viability from FFPE tissues of lung cancer.
- To start fixing the desired slice, carefully transfer it using forceps into a 10 centimeter plate containing a soaked filter paper filled with 2 to 3 milliliters PBS and let it float. Then lift out the filter paper with a pair of forceps and place it in a histo cassette. Transfer the filter paper into a histo cassette. Add a drop of diluted hematoxylin on top of the tissue slice to mark the position of the slice for the subsequent steps. Close the cassette and transfer it into 4% neutral buffered formalin solution and leave overnight at 4 degrees Celsius. After washing and processing of the cassette as described in the protocol, open the histo cassette and use a scalpel to carefully lift the fixed slice from the filter paper. Discard the filter paper and transfer the slice into a mold containing liquid paraffin.
Use a flat weight to press the tissue against the bottom of the embedding mold to ensure even sectioning. Place the bottom part of the histo cassette on top of the mold, add liquid paraffin on top of it, and let the mold cool on a cold plate for 30 minutes. After that, separate the cooled mold from the paraffin block. Use a microtome to prepare 4 micrometer thin sections of the FFPE tissue slice blocks. Trim away the excess of paraffin surrounding the tissue. To obtain even sections throughout the tissue adjust the angle of the block so that the surface of the block is horizontally oriented with respect to the blade. Using a thin brush, collect sequential tissue sections of the paraffin embedded tissue slice starting at the upper part of the glass slides. Continue collecting the sections of the deeper tissue layers to the middle followed by the bottom part of the glass slides.
- The collection of sections from different layers of each tissue slice to the object slides is done to enable capture of potential culture induced gradients in viability, cell migration, or biomarker expression.
- After collecting the sections, continue with their processing and further analysis as described in the protocol.