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

Overview

The following video describes how to preserve morphology of a tissue with formalin fixation and paraffin embedding.

Protocol

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.

Representative Results

Figure 1: Schematic representation of the workflow for establishment and analysis of murine NSCLC tumor-derived slice explants. (A) Schematic describing the collection and preparation of tumor-bearing lungs for slicing. Lung lobes are harvested from a mouse and tumor tissue is dissected away from normal tissue. The black arrowhead and asterisk indicate approximately 4 mm and 1 mm tumors, respectively. The white arrowhead indicates lung tissue glued to the surface of the specimen holder. The red arrow points at an additional piece of normal lung support tissue to retain the tumor in an upright position. (B) Vibratome slicing and collection of tissue slices. White arrow indicates the slicing direction. Collection of sequential slices into a 24-well plate containing cold HBSS + P/S. The slices can either be cultured for different time points (here, 24–72 h) to assess tumor-specific marker expression during cultivation (top row), or can be used to perform drug treatments. C: vehicle control, T: drug treatment (bottom row). (C) Placing the tissue slice for cultivation using rotating incubation units. Tilt the 6-well plate so that some medium covers the top of the grid, place the tissue slice in the middle of the grid on top of the medium, and spread the slice using forceps. Ensure that the 6-well plates are weight balanced for a smooth rotation cycle. X: indicates incorrect, and ✓: indicates correct positioning of the slice. (D) Photograph of the FFPE block of a tumor slice. Black arrow points at paraffin-embedded tissue slice stained with hematoxylin. (E) Schematics showing the sectioning order of the slices in FFPE blocks; these sections can be processed to assess tissue viability and tumor-specific biomarker expression.

Materials

Name	Company	Catalog Number	Comments
10 cm tissue culture plate	Sarstedt	83.1802
PBS	Lonza	BE17-517Q
Formaldehyde	Fisher	F/1501/PB08
Trifold histo cassette paper	Cancer Diagnostics	DX26280
Histo cassettes	VWR	720-2199
Microtome	Thermo Fischer Scientific	HM355S
Leica VT1200 S vibrating blade microtome	Leica Biosystems	14048142066
Hematoxylin for H&E staining	Merck	1.09249.0500
Hematoxylin for counter staining	Dako	S3309
Eosin	Sigma	E4382
Hank’s Balanced salt solution (HBSS)	Sigma	H6648