The Effect of Washing After Protein Blocking on Immunohistochemical Staining Applied to Paraffin Sections

Abstract

Aim: Immunohistochemistry is still frequently preferred in both diagnostic and experimental studies
 because it can show proteins where they are in the tissue. One of the main problems in immunohistochemistry
 is the background staining, which can be prevented by protein blocking and which occurs as
 a result of binding of primary antibodies to tissue proteins and Fc receptors in the tissue due to antigenic
 similarity. There is no consensus on whether to wash after protein blocking in current publications and
 immunohistochemistry manuals published by manufacturers.
 Material and Methods: In our study, routine immunohistochemistry procedure was applied to determine
 the expression of TNF-α on 5 μm thick sections obtained from rat gastric tissue samples in which an
 experimental gastric ulcer model was created with ethanol, and two groups were formed, with and without
 washing after protein blocking, with 10 sections in each group. For semi-quantitative evaluation, the
 histological score (h-score) was calculated from the images obtained from the immunohistochemically
 stained preparations of both groups and the obtained data were statistically compared.
 Results: As a result of our study, no statistically significant difference was found between the h-score
 values of the groups that were washed after protein blocking in immunohistochemical staining and
 the groups that did not wash (p=0.971). The median (min-max) values of the groups with and without
 washing are 211 (179-244) and 215 (171-251), respectively.
 Conclusion: In the immunohistochemical staining procedure, washing after protein blocking does not
 change the staining intensity, does not create background staining, and does not affect the result of
 the h-score for semiquantitative evaluation. Similar studies are recommended for other species, tissue
 types and antigens.