We are following fluorescein-tagged PMO oligomers in skeletal muscle. It requires amplification with a labelled anti fluorescein antibody and we do not see it for more than 2-3 days.  We have checked to see whether the fluorescein tag makes any difference to the exon-skipping effectiveness, and it doesn't. Biotin labelling did not work for us.

it should not be a problem, but one thing you should check, the pH if you did not see your stuff...

and if you want to check further with more detail and higher resolution with enough structure preservation - the best protocols arround are those from the following two papers:

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei.

Rauch J, Knoch TA, Solovei I, Teller K, Stein S, Buiting K, Horsthemke B, Langowski J, Cremer T, Hausmann M, Cremer C.

Differentiation. 2008 Jan;76(1):66-82. Epub 2007 Nov 26.

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions.

Jhunjhunwala S, van Zelm MC, Peak MM, Cutchin S, Riblet R, van Dongen JJ, Grosveld FG, Knoch TA, Murre C.

Cell. 2008 Apr 18;133(2):265-79. doi: 10.1016/j.cell.2008.03.024.

unfortunately they are from work I did, but I can assure you that this protocols were first implemented in 1996/1997 and since tested dozens of times - so this is still the best around.

if you do not want denaturation, then combo-fish is the way to go, which is a variant of this protocol  - check the papers from Michael Hausmann.

a remark at the end - I have seen nearly all preparations in the last years and can tell you that there is still nothing better then the above including combo-fish = it is also interesting that many people claim doing 3D-fish with improved protocols, but you see very clearly that that are empty claims...

ah and the paper from cremer from above belongs also to our papers, actually our protocol from the above mentioned papers was developed for the project "Light optical precision measurements..." - I know, because I still have the bottle of champagne (empty of course) on my shelf when the protocol worked for the first time properly after the first preparation done by joachim rauch, irina solovei, and myself in munich...

Is it strongly recommended to avoid trypsin treatment when the target is 3D-FISH combined with immunostaining? - ResearchGate. Available from: https://www.researchgate.net/post/Is_it_strongly_recommended_to_avoid_trypsin_treatment_when_the_target_is_3D-FISH_combined_with_immunostaining [accessed Feb 10, 2016].

Many Thanks to both Terry and Tobias for your help.

There is one critical question that's really bothering me and I can't seem to figure it out. As I understand it FISH assays are normally used for targets that won't wash away with the probe. However my Oligo targets are same size as my FISH probe (25-mers), so what is preventing the oligo or even the hybridised oligo+probe from washing away? 

unspecific binding, steric trapping, or naturally the hybridication = so that is, of course, one thing you might need to consider if you did not see it...

but if you want to see how oligos distribute as oligos, so not for FISH, then you should do this in vivo - send me an email, and let's get in contact, but you might consider the following papers for that:

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. Weidemann T, Wachsmuth M, Knoch TA, Müller G, Waldeck W, Langowski J. J Mol Biol. 2003 Nov 21;334(2):229-40.

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Wachsmuth M, Weidemann T, Müller G, Hoffmann-Rohrer UW, Knoch TA, Waldeck W, Langowski J. Biophys J. 2003 May;84(5):3353-63.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin. Tóth KF, Knoch TA, Wachsmuth M, Frank-Stöhr M, Stöhr M, Bacher CP, Müller G, Rippe K. J Cell Sci. 2004 Aug 15;117(Pt 18):4277-87. Epub 2004 Aug 3.

Detection of NGF-receptors TrkA and p75NTR in human tumor cell lines and effect of NGF on the growth characteristic of the UT-7/EPO cell line. Westphal G, van den Berg-Stein S, Braun K, Knoch TA, Dümmerling M, Langowski J, Debus J, Friedrich E. J Exp Clin Cancer Res. 2002 Jun;21(2):255-67.

so and for high resolution stuff and quantification:

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei. Rauch J, Knoch TA, Solovei I, Teller K, Stein S, Buiting K, Horsthemke B, Langowski J, Cremer T, Hausmann M, Cremer C. Differentiation. 2008 Jan;76(1):66-82. Epub 2007 Nov 26.

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Jhunjhunwala S, van Zelm MC, Peak MM, Cutchin S, Riblet R, van Dongen JJ, Grosveld FG, Knoch TA, Murre C. Cell. 2008 Apr 18;133(2):265-79. doi: 10.1016/j.cell.2008.03.024.

Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation. van de Corput MP, de Boer E, Knoch TA, van Cappellen WA, Quintanilla A, Ferrand L, Grosveld FG. J Cell Sci. 2012 Oct 1;125(Pt 19):4630-9. doi: 10.1242/jcs.108522. Epub 2012 Jul 5.