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. In contrast to commonly used fixation reagents, e.g. formaldehyde (FA), glyoxal can be a good alternative for sensitive antigens masked by FA crosslinking. In this case, the animal is perfused with saline or PBS and the tissue is immersion fixed with glyoxal. We use two different glyoxal solutions for fixation, glyoxal solution A: 3% glyoxal, 1% acetic acid, 20% ethanol in ddH2O according to Richter et al. 2017, or glyoxal solution B: 9% glyoxal, 8% acetic acid in ddH2O according to Konno et al. 2023. Glyoxal solution A seems to be very good for antigens located in or close to blood vessels, e.g. the blood brain barrier. Glyoxal solution B appears to be optimal for some critical antigens located in the postsynaptic density. The immersion fixed tissue can be cut at the vibratome after 48 h of fixation, or can be frozen and cut at the cryostat. The tissue is stained according to our standard protocols (staining protocol - free floating or staining protocol - slide mounted).
Vibratome sections
Material and reagents
- Cold 0.9% saline containing 17 U/ml Heparin
- Fixation buffer A: 3% glyoxal, 1% acetic acid, 20% ethanol in ddH2O pH 4.2-4.4, according to Richter et al. 2017.
- Fixation buffer B: 9% glyoxal, 8% acetic acid in ddH2O, pH 4.2-4.4, according to Konno et al. 2023.
- 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.
- After tissue dissection, postfix tissue in fixation buffer A or B at 4°C for 2-14 days.
- Cut tissue earliest 2 days after fixation with a vibratome (50 µm) and store either at 4°C for up to14 days in fixation buffer or for longer storage at -20°C in cryoprotectant buffer until staining procedure (staining protocol - free floating).
Cryostat sections
Materials and reagents
- Cold 0.9% saline containing 17 U/ml Heparin
- Fixation buffer A: 3% glyoxal, 1% acetic acid, 20% ethanol in ddH2O pH 4.2-4.4, according to Richter et al. 2017.
- Fixation buffer B: 9% glyoxal, 8% acetic acid in ddH2O, pH 4.2-4.4, according to Konno et al. 2023.
- 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.
- After tissue dissection, postfix tissue in fixation buffer A or B at 4°C for 2-14 days.
- Incubate tissue in 30% sucrose in PBS pH 7.4 for 1-3 days at 4°C.
- 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 - 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.
