- I am working on Immunohistochemistry for Per1 protein expression on mouse brain coronal sections (40microns thickness, free floating).
- In the protocol that I'm following, it says that the DAB exposure time is 1-30mins and I have observed different people using different exposure time.
- 30mins can cause too much background staining and 5mins barely did anything for my protein staining.
Hello Priyadarshini Dutta
Yes, different people use different exposure time. The developing time is not fixable, and you should control it under the microscope. The background intensity and specific-staining intensity are both determined by conditions of DAB incubation.
The DAB developing time can be either too short (1 min) or long. In case the antibody concentration is high or kept for a longer incubation time, the DAB developing time is short. On the other hand, a long DAB developing time might be a result of a low antibody concentration or too short an incubation time.
Please note that the blocking time may also influence the DAB developing time. A short developing time with a high background is the result of an insufficient blocking, while too long the developing time might be due to a too long blocking time.
Once you get a clear signal of specific staining, washing steps may be performed.
Best.
Starting from values which are known to be good, you can vary time, concentration, and also antibody concentration.
Exposure depends on the concentration of the DAB reagent you are using. Sometimes, the dilution suggested by the manufacturer won't work and it might give lots of background. You could control the reaction in many ways
1. Dilute the DAB in the DAB-buffer more
2. Keep a positive control (Pan CK for example if the tissue has more epithelial cells or a mesenchymal marker (alpha SMA) works too. Time the DAB exposure based on your positive control. 5-10 mins usually works for me.
3. The tissue section is pretty thick. Hence, antigen retrieval should be proper too. If you use HIER, I suggest you to use a pressure cooker method and a basic AR buffer. This could improve the immunoreactivity. If the localisation of your protein of interest is in the nucleus, good antigen retrieval ensures you get good signal at the end
4. If the kit you are using provides a superenhancer solution, consider increasing the incubation time of the same
Taking together: it is highly recommanded to start with a test serie. That means to test different antibody dilutions and different DAB reaction times. It takes time to move several sections from one bath to the next one. 1 min reaction time in DAB is very ambitioned under this circumstance. So take a DAB reaction time which makes it comfortable to work with. You can interrupt the reaction for checking under the microscope any time. A negative control is helpful to get an impression of the unspecific background.
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