Preparation of Formalin-Fixed Paraffin-Embedded Tumor Samples: A Protocol to Preserve Tissue and Cytology Samples from Non-small Cell Lung Cancer (NSCLC)

1. Preparation of Tumor Tissue Samples

1. Fix tumor tissue in 10% neutral buffered formalin (NBF) in a cassette.
   NOTE: Tumor tissue from proximal lung tumors is generally obtained by bronchoscopy, performed under moderate sedation by a pulmonologist, or lung specialist. For peripheral lesions and diffuse lung disease, a transbronchial or needle biopsy is indicated. These procedures are usually done in a surgery room or intensive care unit.
2. Embed the cassette in paraffin.
   NOTE: Fixation can be achieved by perfusion or immersion immediately following dissection, and typically requires 8–10 h. The fixative volume should be 15–20x higher than the specimen volume. It is not recommended to fix biopsy tissue for more than 10 h, because overfixation can cause masking of the antigen. The fixation speed is about 1 mm/h at room temperature.
3. Infiltrate the fixed tissue sample with wax in the tissue processor (see Table of Materials).
   NOTE: Dehydration is performed in three alcohol baths with increasing concentrations 70%, 85%, and 90%. Water is finally removed by three final absolute alcohol baths. The clearing is performed in three toluene baths, and the wax infiltration in hot wax baths (44–60 °C).
4. Embed the tissue inside a mold filled with molten paraffin (see Table of Materials) and wait for solidification.
5. Section the paraffin-embedded tissue at a thickness of 3 μm on a microtome.
6. Transfer the paraffin ribbon to a positively charged microscope glass slide (see Table of Materials).
7. Dry the slide for 1 h at 37 °C.
   Note: Store the tissue sections at 2–8 °C (preferred) or at room temperature up to 25 °C to preserve antigenicity, and stain within 15 d of sectioning.

2. Preparation of Cytology Samples

1. Collect bronchial washings in a preservative solution (see Table of Materials).
   NOTE: For this, standard bronchoscopy technique is used.
   1. Lavage the distribution of the bronchus to be sampled. Collect the wash in a clean container. Label the container with the patient’s first and last name, date of birth, and specimen source.
2. Transfer the bronchial washings to a 50 mL conical tube and add 2 g of DL-Dithiothreitol powder (see Table of Materials), vortex the tube for 30 min, and then centrifuge it at 250 x g for 5 min at room temperature.
3. Remove the supernatant and add 10 mL of a mucolytic solution (see Table of Material).
4. Shake the sample for 20 min at medium speed (level 9) and centrifuge it at 250 x g for 5 min at room temperature.
5. Remove the supernatant and deposit the cell pellet in collection tubes containing 10% NBF.
6. Add 4 drops of a cell block preparation reagent (see Table of Materials) to the cell pellet.
7. Transfer the cell pellet to a cassette, fix it in 10% NBF, and paraffin-embed it for cell block preparation and sectioning (see steps 1.1–1.7).