There are protocols out there for stripping and re-probing tissue and cells. We fiddled with some of these, mostly using cell cultures that had been double or triple immunostained, and had mixed results. In some cases we got elution of the antibodies from the first round of staining, but variable success in immunolabeling a second time. In other cases we could only get partial elution of the first round of antibodies. Nucleic acid labeling with DAPI, etc. is destroyed. We tried a wide range of treatment times from a few seconds up to 3 hours. In many cases there was serious structural damage to the cells. All-in-all it was a pretty negative exercise, and we never found anything that I was satisfied with in terms of complete elution of the first round of antibodies while leaving the sample structurally sound and allowing good detection of the second set of antigens. Mostly what we found was that these treatments made my tech frustrated and angry. Thicker wholemounts or sturdier sections might fare better than cultured cells and the frozen sections we worked with.

Buffer formulations we tried included:

1) KMnO4 +/- detergent -This treatment was closest to working for us, but we were never able never quite able to get the combination of tissue and staining quality we needed.

2) KMnO4 + H2SO4 +/- HCl +/- detergent

3) low pH glycine-HCl buffer +/- detergent +/- microwave treatment;

Some references you might look at for these treatments include:

Bu'lock et al. 1983. J Histochem Cytochem 31: 1356-1362

Espada et al., 2001. Histochem Cell Biol. 116:41-47.

Li et al., 2007. BMC Neurosci 8:22

Lan et al. 1995. J Histochem Cytochem 43: 97-102

Tramu et al. 1978 J Histochem Cytochem 26: 322-324

Wahlby et al., 2001. Cytometry 47:32-41.

There is also at least one protocol out there for elution of antibodies from plastic-embedded sections that has worked beautifully for multiple labeling and array tomography in the hands of the Micheva and colleagues in the Smith lab from Stanford. They have a whole series of protocols published in Cold Spring Harbor Protocols in 2010. Also see:

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.

Micheva KD, Busse B, Weiler NC, O'Rourke N, Smith SJ.

Neuron. 2010 Nov 18;68(4):639-53. doi: 10.1016/j.neuron.2010.09.024.

PMID: 21092855

Hope any of this helps and that it works better for you than me.....

This may be a bit late, but I'm wondering if you (or anyone) has tried the simple protocol found here (Ranjan 2012, doi:10.1016/j.jim.2012.02.018)?  It is just Tris buffer, pH 6.8 with 2% SDS and 20 mM DTT, incubate a few hours at RT.

I would also like to reprobe some samples (cell culture, fixed with 4% PFA and/or 2% Glutaraldehyde), and would appreciate any advice/experiences that people may have. 

Justin did you ever try the 2% SDS Tris buffer?

Justin, I'm sorry I missed responding to your question for over a year! I have not tried any protocols that include DTT.

However, I have successfully stripped and reprobed cultured cells (smooth muscle and HeLa) grown on coverslips and fixed with 4% paraformaldehyde using the the stripping buffer described by Micheva et al. 2010 (ref below). In those cells we labeled endosomes for Rab5 (rabbit primary Ab), imaged, stripped and reprobed for Rab7 (also a rabbit primary Ab) and imaged the same field of cells. The Rab5 and Rab7 antibodies labeled different populations of endosomes as one would expect.

  We deviated from the Micheva procedure (which was developed for plastic sections and calls for drying the sections between staining runs) slightly. After imaging Rab 5 labeling, we floated the coverslips off of the slides they were mounted on by immersing in warm PBS for about 30-45 to dissolve the Prolong Gold we used to mount the coverslip. The coverslips were rinsed several times in PBS to be certain that all the Prolong Gold was gone and then we performed the stripping step using the Micheva et al stripping buffer and rinsed several times in PBS, blocked non-specific labeling and then followed our standard  immunolabeling protocol using the second primary antibody against Rab7. 

   I have not tried the stripping and reprobing procedure on cells fixed with glutaraldehyde, but glutaraldehyde itself  will create enormous autofluorescence in the green channel. You can reduce this with a short (1-2 minute) incubation of your cells in 1% NaBH4 dissolved in ddH2O for 1-2 min at room temp (DO NOT USE PBS or HBSS!!! for this step- it will destroy your cells), rinse 3x quickly in ddH2O, exchange to PBS or HBSS and start your immunolabeling.

  Hope this isn't too late to be useful. Good luck

Dave Sherry

The Reference for the stripping buffer is:

Micheva KD, Busse B, Weiler NC, O'Rourke N, Smith SJ.

Neuron. 2010 Nov 18;68(4):639-53. doi: 10.1016/j.neuron.2010.09.024.

PMID: 21092855 

The Cold Spring Harbor series of papers (2010) by these authors has a detailed description of the stripping buffer recipe and the procedure Micheva et al. used for stripping and reprobing plastic sections. (put in Micheva KD into Pubmed and the links will come up)

Thanks David!

We were able to develop a method, though we don't strip, we darken the probes. We have done this on tissue as well.

I have tried the DTT method on whole mounted mouse tissue and it works well for some antibodies, but not others. In general, for second and third round staining, nuclear antibodies don't seem to stick after the treatment, but anything in the cytosol or in the membrane (so far) seems to work well. So we generally perform immunostaining for any nuclear proteins first and then strip and re-probe, and then label cytosolic proteins. Also, some antibodies hang around better than others so we have just started doing 20mM DTT overnight rather than the 3 hours recommended in the Ranjan et al. paper. Finally, I will say that we work in the cochlea where a whole-mounted organ of corti is only a few cell layers thick, so it is almost like working in sections. I don't know how well this would work with other whole-mounted tissues if they are thicker than 50-100 um.

Justin or others, has anybody tried the methods mentioned above with any success? I want to double stain MCF-7 cells grown on a glass coverslip, have primary antibodies of different origin and corresponding secondary antibodies conjugated to HRP. I want to visualize with HRP, strip and re-probe and then overlay images of the same fields. I am looking for the best way to strip the first round of antibodies (or at least the secondary)