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Standard Protocols for Tissue Preparation for IHC

Tissue quality is crucial for the success of immunohistochemical experiments. Complete perfusion is important to remove residual IgG containing blood from blood vessels. These IgGs may be bound by cross-reactive secondary reagents and cause undesired background staining. Too weak or too strong fixation may lead to bad tissue integrity or the masking of antibody binding epitopes. 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 can be determined individually.

Tissue preparation for vibratome sections

Solutions needed

  • 0.9% saline
  • Fixation solution: 4% paraformaldehyde, 50 mM phosphate buffer, pH 7.2, 150 mM NaCl
  • 0.1 M Tris-HCl, pH 7.2
  • TBS: 20 mM Tris, pH 7.2, 150 mM NaCl
  • Cryoprotectant solution: 30% sucrose, 1% Polyvinyl-pyrrolidone (PVP-40), 30% ethylene glycol, 50 mM phosphate buffer pH 7.2

Procedure

  1. Transcardially perfuse with 0.9% saline for 1 min.
  2. Perfuse with fixation solution for 10 - 20 min with a rate of 12 ml/min.
  3. Postfix tissue in fixation solution for 24 h at 4°C.
  4. Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer three times.
  5. Cut tissue (30 - 50 µm) with a vibratome and keep in TBS.
  6. Continue with staining procedure.
  7. For long term storage keep sections at -20°C in cryoprotectant solution.

Tissue preparation for cryo sections

Solutions needed

  • 0.9% saline
  • Fixation solution: 4% paraformaldehyde, 50 mM phosphate buffer, pH 7.2, 150 mM NaCl
  • 0.1 M Tris-HCl, pH 7.2
  • TBS: 20 mM Tris, pH 7.2, 150 mM NaCl
  • 20% sucrose in 0,1 M Tris-HCl, pH 7.2
  • Embedding medium for cryostat sectioning

Procedure

  1. Transcardially perfuse with 0.9% saline for 1 min.
  2. Perfuse with fixation solution for 10 - 20 min with a rate of 12 ml/min.
  3. Fix tissue in fixation solution for 24 h at 4°C.
  4. Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer three times.
  5. Incubate tissue for 12 - 18 h in 20% sucrose, 0.1 M Tris-HCl, pH 7.2.
  6. Freeze tissue.
  7. For long term storage keep tissue at -80°C.
  8. Cut tissue (6 - 10 µm) and mount sections on slides. Optimal section thickness and cutting temperature can vary between tissues.
  9. Allow frozen sections to dry for 20 - 30 min at RT.
  10. Continue with staining procedure.
 

Tissue preparation with mild, low pH fixation

Solutions needed

  • 0.9% saline
  • Fixation solution: 1 - 2% paraformaldehyde, 100 mM Na-acetate buffer, pH 6.0
  • Phosphate buffer: 100 mM phosphate buffer, pH 7.3
  • TBS: 20 mM Tris, pH 7.3, 150 mM NaCl

Procedure

  1. Transcardially perfuse with 0.9% saline for 1 min.
  2. Perfuse with fixation solution for 10 - 20 min with a rate of 12 ml/min.
  3. Cut tissue (60 µm) with a vibratome. The tissue is very fragile. This step has to be carried out very carefully.
  4. Wash in phosphate buffer for 10 min.
  5. Wash in TBS for 10 min
  6. Directly proceed to the staining procedure. This weakly fixed tissue cannot be stored.

Tissue preparation with paraffin-embedding (FFPE)

Solutions needed

  • 0.9% saline
  • Fixation solution: 4% paraformaldehyde, 50 mM phosphate buffer, pH 7.2, 150 mM NaCl
  • 0.1 M Tris-HCl, pH 7.2
  • TBS: 20 mM Tris, pH 7.2, 150 mM NaCl
  • 70% ethanol
  • 90% ethanol
  • 100% ethanol
  • Xylol
  • Paraffin

Procedure

  1. Transcardially perfuse with 0.9% saline for 1 min.
  2. Perfuse with fixation solution for 10 - 20 min with a rate of 12 ml/min.
  3. Postfix tissue in fixation solution for 24 h at 4°C.
  4. Transfer tissue to 70% ethanol and incubate for 1 h at 40°C.
  5. Transfer tissue to 90% ethanol and incubate for 1 h at 40°C.
  6. Repeat step 5 with fresh 90% etahnol.
  7. Transfer tissue to 100% ethanol and incubate for 1 h at 40°C.
  8. Repeat step 7 twice with fresh 100% ethanol.
  9. Transfer tissue to xylol and incubate for 1 h at 40°C.
  10. Repeat step 9 with fresh xylol.
  11. Transfer tissue to paraffin and incubate for 45 min at 63°C.
  12. Repeat step 11 three times with fresh paraffin.
  13. After cooling down the tissue can be cut with a microtome.
  14. Continue with staining procedure for chromogenic or fluorescent detection.