Publication protocol
Specimens obtained during surgery were quickly fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin using standard techniques. Paraffin-embedded samples were serially sectioned at 4 μm, mounted on adhesion microscope slides, and baked at 60 °C for 1 h. After paraffin sections were deparaffinized in xylene and hydrated through a graded series of alcohol, they were treated in 10 mM (pH 6.0) citrate buffer for 5 min by autoclaving to retrieve antigenicity, cooled to room temperature, blocked with 3% H2O2 for 10 min, and rinsed 3 times for 3 min in phosphate-buffered saline (PBS, pH 7.2–7.4). The sections were then incubated with 10% normal goat serum in PBS to block nonspecific protein binding, followed by incubation with rabbit anti-human IgG polyclonal anti-Reg IV (Bioss, Beijing, China) at a 1:300 dilution at 37 °C for 2.5 h, and washed with PBS. The Reg IV signals were amplified and visualized with a biotinylated secondary antibody at 37 °C for 20 min and a horseradish peroxidase polymer conjugate at 37 °C for 15 min following the protocol in SP-9000 Histostain™ Plus Kits (ZYMED, South San Francisco, CA, USA). Finally, sections were stained for 5–10 min with 3–3′-Diaminobenzidine (DAB) and counterstained with 0.1% hematoxylin for nuclear staining. A negative control was set simultaneously by replacing the primary antibody with PBS. Two independent investigators analyzed samples, and differences in scoring were discussed until consensus was reached. For evaluating Reg IV expression in the various samples, a scoring criterion was taken from Sinicrope et al. [25]. For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. A mean percentage of positive tumor cells was determined in at least 5 areas at × 100 magnification and assigned to one of the 5 following categories: (a) 0, < 5%; (b) 1, 5–25%; (c) 2, 25–50%; (d) 3, 50–75%; and (e) 4, > 75%. The intensity of Reg IV immunostaining was scored as follows: (a) weak, 1+; (b) moderate, 2+; and (c) intense, 3+. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. Cases with weighted scores of less than 3 were defined as negative; otherwise they were defined as positive. Cytoplasm staining was defined as a positive expression for Reg IV. For SOX9 (1:300, Bioss) staining, the procedure used was as described above. Only nuclear immunoreactivity was considered as a positive expression.
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