Hi all, I am doing IHC to check gene localization in liver tissue, but every time the color of DAB very dark drown and hematoxylin color is faded in my result. I want to it is normal color of IHC or doing some mistakes, like the time of DAB application is too long and I need to cut the time duration from 5 minutes to 1 minute. if some other technique available to decrease DAB color to show good background then kindly help me out.
Attached file contain my results, tell me it is normal for liver tissue or need to do something to make my results better. Thanks in advance.
Please give us your protocol in detail to see what fixation, pretreatment, detections-system you use. In IHC there are so many points, where something can go wrong. Mostly too much "brown" has it's cause anywhere in the procedure. e.g. endogenous enzyme, endogenous biotin..
Dear Haseeb,
basically, the liver tissue is super sensitive in DAB exposure and you should decrease the exposure duration as much as you can, also sometimes in the higher concentration of primary Ab you will come up with higher brown density.
the other point is sodium citrate boiling step (less than 2 min when its bubbling) which should be done at pH=6 and 15min in steamer.
these are all came in my mind.
@ Gudrun Lang I used following protocol with some major steps
1. Deparaffinize the slides
2. Citrate antigen retrieval buffer and microwave (700 watt oven) for 5 minute at maximum heat and 20 minute at minimum heat and let the solution cool down at room temp
3. Block endogenous peroxidase activity with 3% H202 for 10 minutes
4. 5% goat serum for 30 minutes at room temperature
5. Primary antibody made in TBS (1:100 and 1:200 both try) for overnight at 4 centigrade
6. HRP for 30 minutes at 37 C
7. DAB for 2-3 minutes
Wash the sections with TBS solution after each step and Counterstain in Mayors Hematoxylin for 2 minutes
step 2: decrease to less than 2 min oven and 15 min steamer
step 5: Prepare Primary Antibody: (in PBSor TBS + 2% serum)
Step 6: 1hour
DAB: 30s-1.5min
Do you use a polymer-detection-system, where the secondary is conjugated directly to the peroxidase? Or differently asked: Is your detection-system biotin-free?
Should your antigen be stained in the nucleus or in cytoplasma?
Is the 3% peroxidase fresh?
What I see in the picture is a rather clear stain confined to hepatocytes. The surrounding connective-tissue cells are unstained or stained with hematoxylin. So in my opinion it is not an effect of overstaining, rather a kind of unspecific stain in the hepatocytes due to endogenous biotin or peroxidase. Endogenous biotin is likely to be seen in nuclei (correct me if I am wrong). A short google-search give me the impression, that liver-nuclei contain a biotin-binding protein. So if you use a biotin-conjugated primary, the antibody would be bound unspecifically in the nuclei.
What about the negative control without any primary?
1. Check the DAB color strength under the microscope.
2. My experience - Stop the DAB reaction in distilled water. Distilled water builds up the reaction. In the PBS buffer, DAB still responds.
Hi Haseeb Khaliq, I think the problem is with peroxidase quenching. Liver is known to have very high levels of peroxidase. You need to increase the your endogenous peroxidase quenching in terms of concentration of hydrogen peroxide as well as as time of exposure to around 30 - 45 min. If you standardize this step, I think you will get good results with liver samples.
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