"Hello,
I am a Ph.D. student at the Gwangju Institute of Science and Technology in South Korea, and I have some questions regarding my immunofluorescence (IF) experiment.
1.High background:
I have noticed that my IF image exhibits a high background, as there is significant staining in the broader areas of the cell. I suspect this may be due to various factors such as high concentration of Ab and problem about secondary Ab.
2. Non-specific staining:
I have observed staining in conditions where cells are not expected to be stained, which leads me to believe that there may be non-specific staining occurring. Is this stained? I am unsure if my troubleshooting approach is correct in addressing this issue.
As this is my first time conducting an IF experiment, I would greatly appreciate any guidance or advice on how to address these challenges effectively.
Thank you.
Hi Donghoon Kang
High background and non-specific staining may be due to many factors such as:
* High antibody dilution or polyclonal nature.
* Blocking Inefficiency
* Washing Ineffectiveness
Can you share your detailed protocol? So we can check if any of the steps need to be optimized.
Thanks,
Thank you for your comment.
Is it high background?
Protocol is below.
Fixation & Permeabilization
The cells may be fixed using one of two methods:
1. Using formalin solution, neutral buffered, 10%(sigma) for 10 min at room temperature
2. Incubating the cells in 100% methanol (chilled at -20°C) at 4°C for 10 min
The cells should be washed three times with ice-cold PBS.
-Blocking and immunostaining
1. Incubate cells with 1% BSA, 22.52 mg/mL glycine in PBS for 30 min to block unspecific binding of the antibodies.
2. Incubate cells in the diluted antibody(1:30 dilution, because I can't see anything when I diluted 1:100 dilution) in 1% BSA in PBS in a humidified chamber for overnight at 4°C.
3. Decant the solution and wash the cells three times in PBS, 5 min each wash.
4. Incubate cells with the secondary antibody(1:1000 dilution) in 1% BSA for 1 h 30 min at room temperature in the dark.
5. Decant the secondary antibody solution and wash three times with PBS for 5 min each in the dark.
Thank you.
Hello Donghoon Kang
Yes, the background appears to be quite high. I would suggest you to include permeabilization in your protocol and block using NGS instead of BSA and wash with 0.1% T-X100 in PBS after every step for 5mins, that would reduce the background and non specific staining.
* Fix with 4% ice cold PFA for 10-15mins in room temp.
* Permeabilize using 0.1% Triton-X-100 for 10mins.
* Blocking using 5% NGS for 1hr in room temp.
* Antibody incubation overnight at 4 degree.
* Secondary antibody Alexa flour for 1hr. Wash 2 times after primary and secondary antibody incubation with 0.1% T-X100 for 10mins.
* Mount and store in 4 degree refrigerator.
Hope it helps,
Thanks,
Hello, Samir Ranjan Panda
Thank you for your prompt reply.
I will try this solution next IF experiment.
I appreciate your reply.
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