This study investigates how different decalcification reagents, including formic acid/formalin and an EDTA-based, influence antigenicity in immunohistochemical (IHC) staining. The analysis was conducted using the CD3 and CD5 antibodies on porcine tissue to evaluate the reagents' effects on staining intensity and background staining. An automated IHC staining apparatus with tha DAB method was used to ensure consistent staining conditions across samples.
The results showed that both reagents effectively preserved antigenicity with strong and comparable staining intensity. However, differences in background staining were observed. The positive control, performed on human tissue, showed no background staining. It is important to note that the human control tissue did not contain bone but rather different tissue types on a single section, which had not undergone decalcification. In contrast, porcine tissue, which had been decalcified, exhibited some background staining with formic acid/formalin and slightly higher levels with EDTA
This raises the following questions:
- Why is no background staining observed in the human control tissue, while some background staining occurs in porcine tissue?
- Could this difference be attributed to variations in antibody affinity between the two tissue types?
- What explains the higher background staining with EDTA compared to formic acid/formalin?
I suggest that you try with porcine control tissue, that has not undergone de-calcification (e.g. spleen, lymph node, intestine) to test if it is a species issue or not. Additionally, you can omit the primary antibody to test if the background is due to the secondary antibody/detection kit.
Background can have diverse causes. Immunglobulines are basic glycoproteins, that tend to bind to negative charged tissue components like proteoglycans (cartilage) or other proteins. Acid or alkaline treatment can increase the effect. It also leads to swelling of collagen fibers, that have also an affinity to immunoglobulins. Another cause may be underfixation, because decalcification is a rather rough treatment and calls for sufficient fixation. Decalcified tissue is more sensitive to antigen retrieval. So for me, it is natural, that background is stronger in decalcified tissues.
Try Christian's hint and try to shorten the AR-time or the antibody-titer. This can show, if decalcified tissue needs just a modified protocol for clear staining.
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