IHC-P: Tissue Preparation Protocol FFPE

Tissue preparation and fixation of formalin-fixed paraffin-embedded tissues (FFPE) are important for the success of immunohistochemical experiments. Many tissue processing parameters have been identified to affect IHC-P staining results, e.g. the delay of tissue removal to fixation (cold ischemia), the duration of fixation (see: Influence of Formalin Fixation Duration on Staining Intensity in FFPE Tissues) and the choice of fixative. Paraffin embedded tissues are most often fixed in either 10% (v/v) neutral buffered formalin (FA) or fresh 4% (w/v) formaldehyde solution (PFA) made from paraformaldehyde power. In our standard tissue preparation protocol, we apply a ready-to-use FA solution with a low amount of methanol. Cardiac perfusion of animals with saline or phosphate buffered saline (PBS) removes residual IgG containing blood from blood vessels. This reduced undesired background, when secondary reagents are used, that cross-react with endogenous IgGs, e.g. mouse-on-mouse. In our standard protocol, we apply 24 h 4% FA fixation for standard samples to obtain a good tissue integrity.

Materials and reagents

• Cold 0.9% saline containing 17 U/ml Heparin
• Fixation buffer: 4% FA in PBS, pH 7.2
• 70% ethanol
• 90% ethanol
• 100% ethanol
• Xylol
• Paraffin

Procedure

1. Transcardially perfuse with 30-50 ml (or until the tissue is visible cleared from blood) cold 0.9% saline containing 17 U/ml heparin with a rate of 5 ml/min.
2. Perfuse with 30-50 ml fixation buffer with a rate of 5 ml/min.
3. After tissue dissection, postfix tissue in fixation buffer for 24 h at 4°C.
4. Transfer tissue to 70% ethanol and incubate until paraffin embedding at 4°C.
5. Dehydration, clearing and paraffin infiltration is performed in an automated tissue processing system:
   • 70% ethanol for 1 h
   • 80% ethanol for 1 h
   • 96% ethanol for 1 h
   • 96% ethanol for 1 h
   • 96% ethanol for 1 h
   • 100% ethanol for 1 h
   • 100% ethanol for 1 h
   • Xylene for 1 h
   • Xylene for 1 h
   • Paraffin for 1 h
   • Paraffin for 1 h
   • Paraffin for 1 h
   • Paraffin for 1 h
6. Remove the paraffin infiltrated tissue specimens from the automated tissue processing system and orient them in a metal base mold. Pour with melted paraffin and carefully transfer the mold onto a cool plate.
7. After the paraffin has solidified, the mold can be removed and the FFPE block is ready for sectioning on the microtome.

Cut tissue with a microtome:

1. Cool the FFPE block.
2. Cut tissue into 2.5 to 5 µm sections and place the sections onto a warm water bath.
3. Pick up the sections onto Superfrost plus slides and dry overnight at 37°C.

Note: The SYSY standard protocol generates good staining results in the SYSY labs and may be used as suggestion. However, to achieve the highest specific signal and lowest non-specific background signal, the best tissue preparation conditions (e.g. duration of fixation, dehydration and clearing procedure) must be determined individually.