UPitt TriState SenNet TMC FFPE Specimen Section
John Sembrat, Marta Bueno
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Abstract
To perform imaging analysis of the FFPE blocks were submitted to hematoxylin and eosin stain. Initially FFPE blocks were prepare by the Research Histology Pitt Biospecimen Core and kept at room temperature (preserved from light) until sectioning.
Before start
Monitor the water bath temperature carefully. The temperature will need to be 5 ‑ 9 ˚C below the melting point of the wax. Make sure the water is clean and free of bubbles and section waste (to avoid cross‑contamination).* Make sure the appropriate slides are ready.
Steps
Section Collection
Using a Histo-Quill Pen, hand label all slides with project number and block ID (add type of stain if needed) .
Ensure paraffin block is at room temperature.
Place block into holder of microtome.
Use clean, sharp microtome knife or disposable blade.
Use forceps or brush instead of your fingers to pick up sections or wax fragments from blade or block face.
The knife or blade should be removed from the microtome when the instrument is left unattended or when cleaning the instrument. It can then be grasped with forceps (not fingers) or picked up with the magnet at the end of a brush and safely removed. Used blades should be disposed of appropriately in a “sharps” container.
Set blade clearance to the optimal clearance angle & orient the specimen in the appropriate position.
Cut section to a 5 μm thickness. 5 µm sections
Note: Dip block face into molten paraffin to seal the tissue and allow to cool before storage .
Float paraffin ribbons on deionized H2O (or RNase-free H2O), heated to just below the melting point of the paraffin being used (usually 42°C).
Mount sections on slide by slowly bringing the flat side of slide up from underneath the floating tissue section, so that the tissue section is centered in the membrane window.
Place slide at an angle to allow water to drain and incubate sample at room temperature for 1h 30m 0s.
Slides can now be stored at room temperature for future use.
Proceed with staining protocol (if needed).
