I am trying to stain for a particular protein in brain mouse tissue slices and am seeing a lot of background staining. The tissue was fixed with PFA. I generally block in goat serum for 1-hour at RT, incubate in primary overnight, and incubate in fluorescent secondary for 30-min. I wash with T-PBS between every incubation 3 times, 5 minutes each wash.
Good points from Elizabeth E LeClair
but you could also try reduce epitope retrieval if that is a step you are using and your seeing non-specific antibody staining.
Hi,
For troubleshooting, you can also try changing the blocking solution. Serum could be more antigenic than purified BSA for example and could lead to some background particularly if using polyclonal antibodies.
Good luck!
Alexandru Sasuclarkthis is the protocol I use and seems to work. you can have a try
· Xylene or Histoclear for 2 min.
· Xylene or Histoclear for 2 min.
· Xylene or Histoclear for 2 min.
· 100% EtOH 2 min
· 100% EtOH 2 min
· 100% EtOH 2 min.
· ddH2O 1min
· Antigen retrieval: Bring the sodium citrate (pH 6.0) into the steamer for 30 min. Leave the sodium citrate for ten minutes and then add the slides to the citrate for 20 min.
· Take the slides of the steamer and put them on ice for 5 min to cool down This allows the slides to cool enough so they may be handled, and allows the antigenic site to re-form after being exposed to high temperature.
· Wash slides in TBS three times for 5 min.
- Permeabilize the samples for 10 min with TBS containing 0.2% Triton X-100 . Triton is not appropriate for membrane-associated antigens since it destroys membranes.
- Wash cells in TBS two times for 5 min.
- Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper. Circle the tissue with a pen. Apply Dako blocking buffer onto the sections and incubate in a humidified chamber for 10 min at room Temperature.
- Drain the blocking buffer from the slides
- Apply the appropriately dilute primary antibody onto the sections and incubate in a humidified chamber at 4 C overnight
· Wash slides in TBS two times for 5 min.
- Apply the appropriately dilute secondary antibody onto the sections and incubate in a humidified chamber at Rt for 45 minutes
- Drain the slides and apply DAPI
· Wash slides in TBS two times for 5 min.
· Wash in ddH2O 1min
· Drain off the slides and mount them with Fluormount
Hi
A) Increase the time of retrieval solution like 20 min B) increase the incubation of secondary Ab 1 hr C) reduce the wash duration.
Good luck!
The suggestions from Elizabeth E LeClair is worth trying. If still the issue is not resolved you can try reusing the primary antibody solution. This helps in reducing non specific binding of the antibody and often has less background in immunostaining. Prepare the antibody solution in 0.02% Na Azide to keep its integrity. Recover your primary Ab solution after first use and keep it at 4 deg C and use the same antibody solution for your next immunostaining experiment. This can definitely help reducing background if it is due to non specific binding of primary Ab
Elizabeth E LeClair
has most of them.one more as the last thing you should check it. In laboratory we usually prefer 30 micron for "brain-stem" sections. if you tried out and found the best thickness, i would suggest to check on antibodies (basically change them to a different supplier's solution).and what is the percentage of your triton x-100 because you might be going little harsh on because of that, too.
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