Formalin-Fixed Paraffin Embedded (FFPE) Tissue Preservation: A Method for Studying Tissue Morphology

1. Fixation and Processing of the Tissue Slices

1. Carefully lift the uncultured 0 h reference or cultured slice onto a filter paper soaked in PBS.
   1. To do this, add 2-3 mL of PBS on top of a filter paper placed in a 10 cm plate. Place the slice on top of the filter paper and lift out the filter paper using a pair of forceps.
   2. Transfer the filter paper into a histocassette, and add a drop of diluted hematoxylin (1:1 in deionized water) on top of the tissue slice to visibly mark the position of the slice during the subsequent processing steps (Figure 1D).
   3. Close the cassette, and transfer it into 4% neutral buffered formalin solution. Fix the tissues overnight at 4 °C.
      NOTE: While placing the slice on top of the filter paper, make sure that the top section of the slice is facing upwards; this is necessary to follow the top, middle, and bottom section of a slice during sectioning and analysis as described in step 2.3.
2. The next day, transfer the cassettes into 70% EtOH, and immediately proceed with the paraffin-embedding tissue processing step.
3. Prior to tissue processing, wash the histocasettes in 100% EtOH 2x for 10 min each. In this case, use a microwave station for tissue processing. Select the program used for 1 mm tissue thickness and follow the instructions provided in the manual (https://www.totaltissuediagnostics.com/images/MM073-005_-_KOS__Operator_Manual.pdf).
   NOTE: When using other tissue processing machines, use the program suitable for thin tissue samples.
4. For paraffin-embedding, open a histocassette and use a scalpel to carefully lift the slice from the filter paper. Discard the filter paper, and transfer the slice into a mold containing liquid paraffin.
   1. Press the tissue against the bottom of the embedding mold, for instance with a flat weight, to ensure even sectioning. Place the bottom part of the histocasette on top of the mold, add liquid paraffin on top of it. Let the mold cool on a cold pate for 30 min, and separate the mold from the paraffin block.
      NOTE: As an alternative to horizontal embedding, it is possible to embed the tumor slice vertically by positioning the slice in an upright position. Vertical sections readily permit analysis of gradients in viability or functional marker expression on a single section. Gradient analysis with horizontally-embedded slices requires paraffin sectioning as described in step 2.2.

2. Processing and Analysis of Formalin-fixed and Paraffin-embedded (FFPE) Tissues

1. Prepare 4 µm thin sections of the FFPE tissue slice blocks using a microtome. When sectioning, adjust the angle of the block so that the surface of the block is horizontally oriented with respect to the blade; this is necessary to obtain even sections throughout the tissue.
2. To enable capture of a potential culture-induced viability gradient, cell migration across the slices, or gradients in biomarker expression across cultured slices, collect sections from the top, center and bottom layers of each of the tissue slice on object slides as explained below.
3. Collect sequential tissue sections of the paraffin-embedded tissue slice first on the upper part of the glass slides. Continue collecting the sections of the deeper tissue layers to the middle followed by bottom part of the glass slides (Figure 1E).
   NOTE: To accommodate three sections on a glass slide, trim away the excess of paraffin surrounding the embedded tissue slice.
4. Allow the sections to dry overnight at 37 °C, and proceed with H&E staining or immunohistochemistry as described below.
   NOTE: Loss of antigenicity can occur when FFPE sections are stored at high temperatures or for extended periods of time. Paraffin sections are recommended to be stored at 4 °C, and IHC analyses should be carried out within 6 months after sectioning.
   1. For H&E staining, deparaffinize and rehydrate the paraffin sections as follows: xylene 3x for 5 min, 100% EtOH 3x for 1 min, 96% EtOH 2x for 1 min, 70% EtOH 1 x 1min, and deionized water 2x for 1 min.
   2. Incubate the sections in freshly filtered hematoxylin solution for 10 min, and wash under running tap water for 5 min. Dip the sections in acid alcohol (1% HCl in 70% EtOH) for 2 times, and wash under running tap water for 5 min followed by incubation with 0.5% eosin for 2 min.
   3. Following the eosin step, dehydrate the sections by immersing the slides in alcohol and xylene solutions, as follows: 96% EtOH 2x for 15 s, 100% EtOH 3x for 30 s, xylene 3x for 1 min. Finally, embed the sections in a xylene-based mounting medium.