[Histonet] In situ hybridization on insects: fixing, processing & cuticle softening advice

Bruce Webber bruce.webber <@t> cefe.cnrs.fr
Thu Apr 5 10:38:45 CDT 2007

Hi Histonetters,

I am looking for advice on preparing and processing small (c. 2-3mm
long) adult ants (i.e. with a well-formed chitinous exoskeleton) for in
situ hybridization (ISH) detection of bacteria (using DNA probes to
target RNA).  The ants are transverse sectioned into 3 parts before
fixing to allow greater penetration into each of the 3 main body cavities.

I've trawled the archives for various options and have consulted my
histological mentor, Bruce Abaloz, but after not finding what I was
looking for, I would greatly appreciate advice, opinions and
recommendations from others.  I do promise to post a summary of any
responses together with any methodological tips I found along the way!

The main problem I see is dealing with the cuticle to allow for good
sectioning (5-8um), but using chemicals & methods that don't interfere
with ISH, damage the target RNA, or interfere with the efficiency of the
ISH probes.  My understanding is that the following factors need to be

1) Fixing the tissue: 
The only available material available at this stage was fixed in 2%
gluteraldehyde (12h at 4°C) and then stored (> 3 weeks) in 80% EtOH
(meaning that gut excision is not an option due to brittle material). 
In the future, 4% paraformaldehyde, 10% neutral buffered formalin or
just straight into 70% EtOH (with slightly shorter storage times) could
also be considered as options if people feel that they may be better
(the latter option would certainly increase the availability of material).

2) Softening the cuticle: 
IMHO this is the biggest potential area for chemical damage/interference
with ISH.  Proposals I am aware of that would not be compatible with ISH
due to acidic damage of the RNA are [1] Perenyi's fluid (4:3:3, 10%
nitric acid:100% EtOH:0.05% chromic acid);  [2] Diaphanol (50% glacial
acetic acid saturated with ClO2); and [3] Bouin's fixative (3:1:2,
saturated picric acid:formaldehyde:glacial acetic acid).

However, other suggestions from previous posts which I know very little
about include [4] chloral hydrate processing (after tissue dehydration,
melt equal parts of chloral hydrate & phenol and immerse tissue for up
to 7 days, followed by chloroform as an intermediary agent);  [5]
Mollifex Gurr (glycerol, phenol, acetone and EtOH solution, applied to
the cutting surface of the paraffin block);  [6] Nair (as in the hair
removal cream - thioglycolate salts & calcium hydroxide, between fixing
and processing, soak the tissue for c. 24hrs); and [7] Phenol (after
fixation, soak fixed specimens in 4% phenol (in 80% EtOH) for 24
hours).  Lastly, there is 1% DMSO (added to the fixative), but given
that the ant is chopped into 3 sections, this may not be necessary to
ensure adequate penetration (but does DMSO also improve sectioning?).

3) Processing the tissue: 
The ongoing debate of xylene v histoclear (or histosolve)..... 
Histoclear is probably safer, but does xylene produce better results? 
Has anyone had experience with either on similar invertebrate material? 
Others have suggested using isopropyl alcohol (instead of EtOH) for
dehydration as is tends to have a less hardening effect on the tissue.

4) Material for embedding: 
I've seen it argued that tougher material such as that containing
chitinous exoskeletons benefits from using a harder embedding medium
(e.g. TissuePrep 2 from Fisher) or one with better infiltration (e.g.
Paraplast X-tra) over standard paraffin.  I've also read that Paraplast
Plus (containing DMSO) makes sectioning of cuticles easier.  Any
opinions/recommendations/experience with similar material and any of
these embedding mediums would be appreciated!

Thank you all in advance for your help with this matter (I apologise for
the length of this post!).

Bruce Webber
Centre d'Ecologie Fonctionnelle et Evolutive, CNRS, France

(Key words: cuticle, exoskeleton, chitin, insect, invertebrate, ant, in
situ hybridization, ISH, softening.)

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