On occasion one may have performed an antibody labeling experiment on very rare cell or tissue samples. The antibodies provided very poor labeling with very high background. Is it possible to remove these poorly labeling antibodies (primaries and secondaries) and relabel the sample?
Answer: Yes, there are two options, both options must be gentle to avoid altering sample integrity and the sample must be equilibrated back to a neutral pH prior to the new blocking and labeling steps:
1) Use mild stripping buffers commonly used for stripping Western blots.
Proc Natl Acad Sci U S A. 2002 October 15; 99(21): 13589–13594.
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"The slides were then washed in Mild Antibody Stripping Solution (Chemicon) for 15 min at 4°C. After three 10-min washes in PBS, slides were blocked by incubation with 20% normal goat serum for 30 min at RT. This incubation was followed by incubation with rabbit anti-cyclin A...."
Chemicon (now part of Millipore)
Catalog # 2502 ReBlot™ Plus Mild Antibody Stripping Solution
Examples of other 'mild' stripping buffers that may be used:
("TBST" commonly used for Westerns)
25 mM Tris-HCl, pH 8.0
125 mm NaCl
0.1% Tween 20
2) The other method utilizes buffers commonly used to elute antibodies from affinity-purification columns, i.e. dissociating antibodies from a ligand/antigen in a column format by the use of various elution buffers. For example, 0.1 M to 0.2 M Glycine (pH 2.5) is a common elution buffer to dissociate antibodies off the columns. If you use a buffer like this on your tissue sample, it is very important to re-equilibrate the cell/tissue back to neutral pH for subsequent blocking and labeling.
1) Expose the sample by removing coverslip and mountant from the sample. For SlowFade or other glycerol-based mountants, simple soaking in a buffer (such as PBS) should be sufficient to remove the mounting medium. For ProLong or other aqueous polymer-based mountants, soak slides in a Coplin jar filled with buffer (such as PBS or similar) in the refrigerator. The polymer-mountant will swell and should slough off from the sample - this may take a few hours to overnight soaking in buffer.
2) The slides with the exposed cell/tissue samples may be incubated in either individual plastic slide holder containers filled with Glycine buffer, Coplin jars filled with the Glycine buffer or flood the sample with enough Glycine buffer to cover the entire cell/tissue sample for 1-5 minutes (this is dependent on the stability of the antibody binding to the antigen). One may decant this and perform another soak with the Glycine buffer to ensure that all antibodies have been removed. The Glycine buffer may be applied and incubated at 4oC to 25oC (refrigerated to room temperature), but never heated.
3) Wash repeatedly (3X to 5X) with a neutral buffer (such as PBS, HBSS with pH from 7.2 to 7.6).
4) Prior to adding any new antibody, one may wish to observe whether the sample has been completely stripped of all antibodies and there is no background fluorescence by mounting in buffer (wet mount), coverslip and viewing under a fluorescence microscope. If fluorescence from antibody labeling persists, repeat Steps #2 and #3.
5) After the cell/tissue sample has been equilibrated with neutral buffer, perform your standard Blocking step and continue on with the new antibody labeling.
http://www.cooperlab.wustl.edu see "Lab Methods, Protocols & Operations", then "Affinity Purification of Antibodies."