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