BACKGROUND: Immunohistochemistry needs quality control for detecting a specific antigen. Hence, tissue fixation and tissue processing must be taken into consideration to obtain the optimal immunostaining results.
OBJECTIVES: To evaluate the interaction and effect of fixation time, fixative concentrations and different sites of rat oral epithelium using PanCk and P63 markers on the results of immunohistochemical staining and identifying the optimum values for these variables.
METHODOLOGY: A total of 200 specimens from rat normal oral mucosa of different sites were studied. Both neutral buffered formalin (NBF) concentrations (10% and 5%) were used for fixation of the specimens according to groups: (A) 10% NBF for 1 day, (B) 10% NBF for 7 days, (C) 5% NBF for 1 day and (D) 5% NBF for 7 days. Haematoxylin & Eosin staining and immunohistochemical staining was performed using PanCk and P63. The variables were measured by a specific scoring system and analysed by IBMSPSS 27.
RESULTS: PanCk and P63 at both concentrations showed significant differences between 1 and 7 days of fixation (<0.001). The effect of fixation duration for PanCk and P63 was strong (r statistic>0.5), with higher expression for day 1 in 10% NBF and after 7 days in 5% NBF. For both markers, there were significant differences between 10% and 5% NBF in all study groups (<0.001). The effect of NBF concentration in both markers was strong (r statistic >0.5). The oral sites showed significant differences in P63 for all study groups (<0.001) while PanCk did not. The effect of oral site on immunohistochemical expression was strong for P63 while it was weak for PanCk.
CONCLUSION: There were varying effects of the studied parameters on immunohistochemical staining. The 10% NBF concentration and 1 day fixation time proved the optimal result for both immunohistochemical markers.