But I think I am not being able to permeabilize the nucleus… Any suggestions?
My antibodies are not very good. Do you know of a good control that I could use to set up the protocol, such an antibody against DNA or histones for example that works well for whole animal staining?
Good controls are an antibody to DNA (Chemicon mouse monoclonal antibody MAB030)
or to a cytoskeletal protein (Amersham L9 anti-tubulin; Calbiochem Ab-1 anti-actin; ICN Biochemicals C4
anti-actin)
If your antibody is not that good, and/or the epitope is of low abundance, once you are sure your fixation methods are working there are a few things that can be tried using transgenes. I am assuming the epitope is a protein.
[ol]- Obtain or build a strain that carries the (wild-type) gene on an array. It should increase the signal considerably. A variant of this is to make animals transgenic for a hs promoter driven construct, and stain an hour after a heat shock pulse.
Get a better antibody. If one is not available, engineer a transgene array in which the gene includes an epitope like FLAG for which there are many good commercial antibodies and control strains available.
Once you know you can detect the epitope, use CRISPR/Cas9 to introduce the epitope into your gene using an oligonucleotide.
When you know that you can get an oligo in there, insert the GFP coding region and skip the antibody staining altogether.[/ol]
Or alternatively, just go straight for the GFP knock-in with CRISPR. The recent selection-based approaches described by Dan Dickinson and Adam Norris seem pretty good. Dan’s plasmids include both a FP and a 3xFLAG tag, so you could look for expression of the FP tagged protein or do anti-FLAG immunofluorescence.
What stage are you interested in visualizing? Different antibodies can often work in one fixation method but not another.
Alternatives to Finney-Ruvkun include Bouin’s Fix (with picric acid), freeze-crack followed by a host of options (Methanol, Methanol-Acetone, dimethylformamide…) or preceded by PFA.
As for a good nuclear marker:
Pan-histone MAB052 from Millipore
H3 antibody from Abcam
Snug: Yes I checked that and requested the antibody to DNA, it just arrived and could not try it yet. I could not find though a paper of reference in which they show an immunostaining using this antibody.
Maduro, Sperm or Egg?, JordanWard: Thanks for CRIPR ideas. I will use them in other part of the project where I need CRIPSR. I am sorry though, I should have said that the epitope is not a protein.
Steve Von Stetina: I have tried freeze and crack with several fixation options. I have to try a bit more but to the moment freeze cracking plus Methanol-Acetone fixation gave best results. I am using the anti H3 antibody from Abcam. With this protocol I can visualize nucleus of eggs, gonads cells, and sometimes intestinal cells.
Is there a trick for immunodetection in the nucleus of other cells, such as neurons for example, or is just a matter of luck with the antibodies?