whole animal immunofluorescence/nucleus

Model Organism: Worms Methods & Reagents
1

Hi,

I am trying to do a whole animal immunofluorescence. My target is inside the nucleus. I am using the classical Modified Finney- Ruvkun protocol:

http://www.wormatlas.org/images/finneyruvkun.pdf

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?

Thanks!
Paola

2

Have you read Janet’s awesome immunostaining section on wormbook?

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)
3

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]

Good luck…

4

Re: epitope tagging with CRISPR/CAS9, the Seydoux lab obtained nice results with FLAG (http://www.ncbi.nlm.nih.gov/pubmed/25249454, Figure 3C).

5

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.

6

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

Good luck!

Steve V

7

Thanks a lot to all for your replies!

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?

Thanks once more to all.
Paola