Protocol for immunohistology

Fixation of Tissues

• Tissue sections (up to 5 mm) are fixed in acetone at 4°C for 3 - 4 days, with a daily change of acetone.
• Incubation in isopropanol for 4 - 8 hrs, with one change of isopropanol.
• Incubation in xylol for 2 hrs, with one change of xylol.
• Tissue sections are infiltrated by liquid paraffin for a minimum period of 4 hrs or overnight, followed by preparation of 5 µm paraffin sections.
• Tissue sections are mounted on adhesive microscope slides.

Cryostat Sections

• Cryostat sections are incubated in acetone at -20°C for 2 - 10 min.
• Storage of the cryostat sections at -20°C.

Immunohistological Staining with the Monoclonal Antibody DF-4

• Removal of paraffin by 2 x 5 min incubation in xylol.
• Washing the tissue-sections with:
  1. decreasing alcohol concentration
  2. H2O (dist.)
  3. 15-30 min in H2O2 / methanol to inhibit endogenous peroxidase activity
  4. 2 x 5 min in Tris / HCl-buffer
  (please start the staining of cryostat sections with step 3; the previous steps are for
  paraffin-sections only.)

Immunohistological Staining with the Monoclonal Antibody DF-4 (cont?d)

• 10 min incubation at room temperature (RT) with rat serum (10 % in Tris / HCl; v/v) to block non-specific binding of the antibodies.
• 30 min incubation at RT with ScheBo^® • DF-4 monoclonal antibody (5-10 µg/ml in Tris / HCl /10 % rat serum).
• 2 x 5 min washing with Tris / HCl.
• 30 min incubation at RT with a bridging antibody (rat-anti-mouse IgG; 1:100 dilution v/v in in Tris / HCl /10 % rat serum).
• 2 x 5 min washing with Tris / HCl.
• 2 x 5 min washing with Tris / HCl.
• 5 - 10 min incubation at RT with diaminobenzidine (DAB)-substrate solution.
• Intensive washing with tap-water, followed by a washing step with H2O (dist.).
• Dehydration of the stained tissue sections with increasing alcohol concentrations and a final incubation with xylol.

Solutions and Buffers

• H2O2 / methanol
  - 100 ml methanol
  - 2.5 ml H2O2 (30 % v/v)
• Tris / HCl-buffer
  - 6.1 g Tris[hydroxymethyl]-aminomethane (Tris)
  - 50 ml H2O (dist.)
  - 37 ml HCl (1 n)
  - adjust pH to 7.6
  - add H2O (dist.) to 1000 ml
• Imidazole-buffer
  - 408.6 mg imidazole
  - 60 ml H2O (dist.)
  - 30 ml HCl (0.1 n)
  - adjust pH to 7.1
• DAB-substrate solution
  - 40 mg DAB
  - 80 ml imidazole-buffer
  - adjust pH to 7.1
  - 28 µl H2O2 (30 % v/v)
  - Filtration of the buffer

Please note: The immunohistochemical staining does not work with formalin-fixed tissues.