| | Re: Can I assess tissue monoclonality with immunohistochemistry against G6PD isoenzym
Used polyclonal rabbit anti-yeast G6PD antibody (Sigma; dilution 1:200) according to Straatsburg and Frederiks (Straatsburg IH, Frederiks WM (1997) In situ analysis of ischaemia/reperfusion injury in rat liver studied in three different models. Int J Exp Pathol 78:149–161). Cryostat sections are air-dried (1 hr or overnight), fixed in cold acetone (10 min, 22 °C), and dried again (10 min). Antigens are exposed for 30 min by using a solution of Tris(10 mM), EDTA (5 mM), NaCl (150 mM), 0.25% gelatin, and 0.5% Tween-20 (Sigma) at pH 8.0. Between all further incubation steps, sections were thoroughly rinsed in PBS. The antibody is diluted in PBS containing 0.2% (w/v) bovine serum albumin (BSA) and 1% (v/v) normal rat serum to block nonspecific staining. Sections are incubated for 60 min at RT. After rinsing, sections are incubated for 90 min at RT with horseradish peroxidase-labeled goat anti-rabbit IgG (Dako, Glostrup, Denmark; dilution 1:100). Peroxidase activity is detected by incubating the sections for 10 min in a medium containing 0.5 mg/ml diaminobenzidine (Fluka), 3 mM H2O2, and 50 mM Tris-HCl buffer (pH 7.6).