Tissue preparation and fixation are important for the success of immunohistochemical experiments. First, it is crucial, that the animal is perfused with saline or phosphate buffered saline (PBS) to remove residual IgG containing blood from blood vessels. Otherwise, IgGs may be bound by cross-reactive secondary reagents and cause undesired background staining. Second, the tissue is fixed to maintain tissue integrity. A commonly used fixative is formaldehyde (FA), which is known for good tissue preservation. Scientists use self-made FA fixation solutions produced by dissolving paraformaldehyd (PFA) in PBS, or they apply ready-to-use FA fixation solutions containing different amounts of methanol for stabilization. In our standard tissue preparation protocol, we apply a ready-to-use FA solution with a low amount of methanol. The optimal fixation time varies between minutes to hours, depending on tissue and antibody. Too short or too long fixation times may lead to bad tissue integrity or masking of antigens. In our standard protocol, we apply 24 h 4% FA fixation to obtain a good tissue integrity. However, some antibodies need shorter/milder FA fixation or even methanol or acetone fixation, because the epitope is prone to be masked by FA crosslinking (please check the remarks sections for deviating fixation protocols).
Vibratome sections
Materials and reagents
- Cold 0.9% saline containing 17 U/ml Heparin
- Fixation buffer: 4% FA in PBS pH 7.4 (room temperature)
- TBS: Tris buffered saline, 50 mM Tris pH 7.2/150 mM NaCl
- Cryoprotectant buffer: 25% glycerol, 25% ethylene glycol, 50% PBS pH 7.4
Procedure
- Transcardially perfuse with 30-50 ml cold 0.9% saline containing 17 U/ml Heparin with a rate of 5 ml/min until the tissue is cleared from blood.
- Perfuse with fixation buffer 30-50 ml with a rate of 5 ml/min.
- After tissue dissection, postfix tissue in fixation buffer for 24 h at 4°C.
- Rinse tissue in TBS and incubate tissue in TBS for 24 h at 4°C to stop FA fixation process.
- Cut tissue 25-50 µm with a vibratome and store at -20°C in cryoprotectant buffer until staining (staining protocol - free floating).
Cryostat sections
Materials and reagents
- Cold 0.9% saline containing 17 U/ml Heparin
- Fixation buffer: 4% FA in PBS pH 7.4 (room temperature)
- TBS: Tris buffered saline, 50 mM Tris, pH 7.2, 150 mM NaCl
- Cryoprotectant buffer: 25% glycerol, 25% ethylene glycol, 50% PBS pH 7.4
- Tissue-Tek®
Procedure
- Transcardially perfuse with 30-50 ml cold 0.9% saline containing 17 U/ml Heparin with a rate of 5 ml/min until the tissue is cleared from blood.
- Perfuse with fixation buffer 30-50 ml with a rate of 5 ml/min.
- After tissue dissection, postfix tissue in fixation buffer for 24 h at 4°C.
- Rinse tissue in TBS and incubate tissue in TBS for 24 h at 4°C to stop FA fixation process.
- Incubate tissue in 30% sucrose in PBS pH 7.4 for 1-3 days.
- Freeze tissue on dry ice and store at -80°C until cutting.
- Cut tissue with a cryostat:
- Sections on slides: Cut tissue 10-20 µm and mount on slides. Dry sections for 30 min at 57°C. Store slides at -20°C until staining procedure (staining protocol - slide mounted).
- Sections free floating: Cut tissue 25-50 µm with a cryostat and store in cryoprotectant buffer at -20°C until staining procedure (staining protocol - free floating).
The SYSY - standard protocols generate good staining results in the SYSY labs. However, to achieve the highest specific signal and lowest non-specific background signal, the best tissue preparation protocol should be determined individually.
