Protokoll zur Immunhistologie

Hier stellen wir das Protokoll zur Immunhistologie zur Verfügung.

Immunohistochemical Staining of Pyruvate Kinase with
the Monoclonal Antibody ScheBo® • DF-4

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.
  • <30 min incubation at RT with a peroxidase-mouse-anti-peroxidase (PAP) complex (1:200 dilution v/v in in Tris / HCl /10 % rat serum).
  • 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