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

• 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 mm paraffin sections
   
• Tissue sections are mounted on adhesive microscope slides.

• Cryostate sections are incubated in acetone at -20°C for 2 - 10 min
   
• Storage of the cryostate sections at -20°C

• 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.)

• 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 mg/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.