[Histonet] Re: Need help with ISH

[Johnson, Teri]

Naira,

There is a lot of information that we need to know before we can really
help you with this.

Why are you using a tyramide amplification system? Because your target
has known low expression? Or because that's what you saw in a paper
somewhere? Very high background staining is a usual result from using
amplification. Do you actually need amplification? Don't use it if you
don't need it.

When doing ISH on cells, the formalin fixed cells will need to be
permeabilized prior to doing any intracellular staining. It's possible
that you have permeabilized the cellular membrane with methanol and
acetone. I prefer using detergent solutions to do this, with either
Saponin or Triton X-100.

It looks like you might be able to simplify your protocol depending on
what you are trying to demonstrate. Regardless, unless you can get
absolutely negative results with no probe or no antibody, your test
samples are a risk for misinterpretation.

A typical protocol looks something like this:

Cells are fixed, then washed in buffer.
Permeabilized with 0.3% Triton x-100 15 minutes.
Enzyme digestion (pronase, proteinase K) improves penetration and
removes associated proteins.
Stabilize cells with a post-fix in 4% PFA in PBS for 5 minutes
Optional - Acetylation with acetic anhydride in TEA buffer
Pre-hybe in hybridization buffer (no probe)
Hybridization in hybe buffer containing probe (time and temperature
determined by probe type and composition)
Stringency washes to remove unbound probes (concentration and
temperature of SSC determined by degree of stringency needed)
Detection system depends on what the probe label is - streptavidin AP or
HRP if probe is biotinylated, or protein block followed by anti-dig
antibody (labeled with AP or HRP) if probe is labeled with digoxigenin.
Appropriate chromogen.

Not mentioned above are the buffer rinses between steps (no rinse
between pre-hybe and probe hybridization).

If you follow a plan such as above and have no background (low noise)
and strong signal, then you need no further amplification.

I recommend reading as much as you can about the ISH process to help you
understand what each step in the protocol does. Good luck!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110