Re: [Histonet] Help with ISH on ants - last request!

From:pruegg@ihctech.net



Bruce,
I have been working on doing IHC in glycol methacrylate processed tissue
(GMA) which might be of use to you, we have even done some inblock
labeling of wet tissue and then processed it into GMA which is much harder
than paraffin, we used it to cut undecalcified bone.  It can be very
difficult to get large molecule IHC/ISh reagents into GMA sections as it
cannot be removed before staining, but if you do the labeling in the wet
tissue you see the signal already there when you cut the section, another
option would be to use methyl methacrylate (MMA) which can be removed
before staining.  I am out of town but contact me directly for some
protocols if you are interested in trying GMA processing for this.
Patsy

> My last request for help!  Please!   I am still yet to receive any
> responses on any of the issues outlined below....  even advice on only
> small parts of the points mentioned would be most appreciated.
>
> Cheers, Bruce
>
>
> On 05/04/2007 16:38, Bruce Webber wrote:
> 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
> addressed:
>
> 1) Fixing the tissue:
> The only available material available at this stage was fixed in 2%
> gluteraldehyde (12h at 4C) 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 on
> tissue for ISH? 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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>



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