Hi all, I’m working on immunohistochemistry for c-fos in rat uterus (1:75). In previous trials with 1:50 and EDTA retrieval, I saw strong non-specific binding. Following advice, I switched to citrate buffer antigen retrieval and diluted the antibody to 1:75, but now I don’t see any clear expression signal.
Protocol summary:
• Dewaxing & rehydration: Standard xylene + graded ethanol → water.
• Antigen retrieval: Microwave in citrate buffer (2 cycles, 8 min each).
• Blocking: Goat serum, 37 °C, 45 min.
• Primary antibody: c-fos (1:75), 4 °C overnight.
• Secondary antibody: 37 °C, 1 h.
• DAB staining: 7 min, followed by hematoxylin counterstain, dehydration, and mounting.
With this protocol, the background is reduced, but c-fos signal is not detectable.
What steps would be recommended to balance reducing non-specific staining while still maintaining detectable c-fos expression? Thanks in advance!
Sodium citrate's repair capacity is too weak for this product. After comprehensive evaluation with R&D, we recommend using EDTA repair (EDTA 1:50; nuclear localization is still clear, though there is some high background, which is still distinguishable). Then, lower the antibody concentration to 1:75. If possible, try using several different concentrations to find the most suitable one.
The answer to this question is provided by MedChemExpress Technical Support. [email protected]
Hello Fan Janice
You may use heat-induced epitope retrieval (HIER) with an alkaline EDTA buffer (pH 8.0–9.0) which is an effective method for c-fos staining in FFPE rat uterus tissue. The high-pH EDTA buffer is generally more efficient than lower-pH citrate buffers for nuclear proteins like c-fos. Though aggressive, it requires careful optimization to achieve the best balance between signal and tissue preservation.
A typical pH 9.0 Tris-EDTA recipe is
10 mM Tris Base,
1 mM EDTA, and
0.05% Tween 20.
You may test different pH levels. For c-fos, an alkaline buffer (pH 9.0) is often most effective. Consider testing a range from pH 8.0 to 9.0 to find the best conditions for your specific antibody. You may also vary the incubation time. If the initial staining is weak, try extending the heating time in 5-minute increments. If there is high background staining or tissue damage, shorten the retrieval time. For more aggressive retrieval, you may use a pressure cooker, which can achieve optimal results in a shorter time and with higher temperatures.
Best,
- Badges
- Science topic
- Similar topics
- Mathematics
- Algebra
- Matrix