When I buy antibodies, I always wonder whether it is necessary to distinguish different dissolution schemes in immunohistochemistry (IHC) and immunofluorescence (IF) applications, and whether it will affect the test results.
1. For example, what buffers are usually used to dissolve primary antibodies/secondary antibodies used for IHC and IF? (PBS, TBS, solutions containing BSA or sodium azide?)
2. Are there differences in incubation times for different experimental purposes? For example, room temperature incubation vs. 4℃ overnight in IHC?
3. Is light-proof incubation a necessary condition in IF experiments?
I hope teachers with relevant experience can share common schemes or precautions. Thank you for your guidance!
[Troubleshooting Questions are selected from MCE customer consultation emails.]
Hi Lucien,
if you buy antibodies commercially they are often dissolved in PBS (pH 7,4 because this is normally the optimal pH for antibodies).
Sodium azide is often added as this inihibts the growth of microorganism and thus improves durability of the antibody. This should be independent from your downstream application (IHC and IF).
The incubation time and temperature (as well as the working solution) depends more on the antibody you use and usually is not dependend on the downstream application (IHC or IF). Usually you should follow the manual you get from the provider, however you can try things out especially regarding incubation time and washing steps (as a rule of thumb the incubation time is shorter if you have higher temperatures, however lower temperatures and longer incubation times might generate less background staininng)
What is also very important for IHC and IF is if you use a secondary antibody or if your primary antibody is already coupled to a fluorochrome or something like HRP. If you have a secondary antibody you have an additional amplification step and you can ususally use less of the primary antibody, however the use of an secondary antibody might contribute to background staining.
So in total it is more import what antibody you have than your downstream application (not if you are doing an IHC or IF)
However what is really important when you are doing IF - avoid light during the use of the fluorescent antibody because light might destroy the fluorophore. So light-proof incubation is necessary as soon as you are using an antibody coupled to a fluorophor.
I hope this helps
1. Recommended antibody dissolution buffer
Immunofluorescence (IF): It is recommended to use phosphate buffered saline (PBST) containing 0.05% Tween-20 to dilute antibodies;
Immunohistochemistry (IHC), flow cytometry (Flow Cytometry): Use phosphate buffered saline (PBS) to dilute antibodies;
Western blotting (WB): Use Tris buffered saline (TBST) containing 0.1% Tween-20 to dissolve antibodies.
2. Recommended antibody incubation time
Primary antibody incubation:
Standard protocol: Overnight incubation at 4°C (12-16 h) can maximize antibody binding efficiency;
Short-term protocol (alternative option): If you need to shorten the time, you can try incubating at 37°C for 1-2 hours, but the signal strength needs to be verified in a preliminary experiment.
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