[Histonet] Tracheal Whole Mounts-long reply
koellingr <@t> comcast.net
koellingr <@t> comcast.net
Mon Jul 16 00:09:46 CDT 2007
A long reply at specifically targeted subject in case you are not interested you are notified.
John/Kim/other interested parties,
I'll give it a shot. With these 2 preamble pointers.
1) I've never done or even seen this with rat trachea so I'm at a loss how mouse and rat compare.
2) While I know a bit about immunology and IHC and consider myself a good anatomist with good dexterity, we had some people with incredible fine skills where I used to work that put me to shame. (Aside-we had someone who was working on ischemia projects and could tie off any part of a mouse aortic arch or put a ligation into the right common carotid, left common carotid or left subclavian, at will and at any part of the vessel, while the mouse was under anesthesia and do it perfectly mouse after mouse-and those are incredibly thin and tiny structures).
Mouse whole mount tracheas: We used very, very small, sharp instruments. Very tiny, thin scissors. Very thin pins (I believe they are used by entomologists for delicate work). Etc. Open, with scissors, the trachea along the ventral mid-line and mount with the mucosa up. It is true you have (6 or 8????) curved tracheal rings to deal with. Again I've never done this with rat and I'm not sure if it is intuitive or counter-intuitive but those "springy curved rings" aren't too difficult to deal with in mouse. Maybe in rat they are thicker and springier and tighter to "flatten"???? Yet they do pose a slight problem. We used plates that were coated with Sylgard (Dow chemical silicone elastomer). Is 2 part liquid that sets up to hard silicon base after curing and perfect for holding pins tight and not wobbling. Can be poured to any thickness you want. I believe it comes in black also if you want that for color contrast in photos. So don't pin one edge with pin at 90 degrees.
The tissue can just "spring" back up the pin from the hole in the tissue. Pin an edge, at a very acute angle (10 degrees), but do so by going across the tissue into the opposite edge and the silicone so that the pin is pinned but lying tight agross the tissue. Do so near a ring. At next ring, do same thing but in opposite direction. That is point the pin in opposite direction, go acroos tissue, pin into edge and into hard silicone so that that pin is lying tight down across the trachea. Don't need to do every ring but in end you have the trachea down flat (it is not mathematically and perfectly flat) with every other thin pin in opposite directions and each pin lying tight across the trachea but actually pinned down on a severe slant. Then fix in the plate / tissue according to needs. Formalin or in our case we used zinc/tris, fixed pins and all. We used zinc/tris as we were after dendritic cells/immune cells in the mucosa from mouse allergy models and could use all the nor
mal flow antibodies and not worry about formalin sensitive epitopes and nasty retrievals. Another reason was that Thibaut that I mentioned was actually in a Belgian group that developed and marketed some sort of "Immunofix" and "immunowax" in Europe. Gayle Callis asked me about those products a while back. I told her and will still give odds that their immunofix was (is) zinc/tris that Beckstead/Nitta developed and BD sells. I got some of their "immunofix". Comapred to my homemade zinc/tris was same pH, same osmolarity, same severe temperature/pH gradient shift characteristic of Tris buffers and precipitated phosphate ions from PBS, presumably as zinc phosphate. I think Thibauts magic Immunofix is really zinc/tris but at 10,000x the true component cost. So back to the whole mounts, just left them pinned and worked on them like that, permealizing with Triton but I'm sure there are many other ways. After immunos would mount on a slide, permount , coverslip and a clamp to hol
d coverslip flat till things dried. A second attack that I took was to cut 2 extremely thin pieces of silicon gasket. Then take out esophagous, cut long as before and using some very, very small alligator clips (electronic gear), clip the esophagous to a slide, mucosa up, a thin strip of silicone gasket at one extreme edge of "laid open esophagous" and clipped to the slide. Same thing on other side. So you had the esophagous flat, mucosa up but each edge under a strip og silicone gasket and from alligator clipped flat. Very edges useless but entire rest of structure exposed. Then fix and stain. In short, this was not something condusive to an SOP. Was part Rube Goldberg, part witchcraft, part luck, sweat, hopes and thinking. Certainly wasn't pretty looking but it did work. See Chiang Chieng-Hsun, Quantitative study of the ganglion neurons of mouse trachea, Cell and Tissue Research 246:2 Nov. 1986. Also James D Moffatt Role of epithelium and acetylcholine in mediating t
he contraction of 5_HT in mouse isolated trachea. British Journal of Pharmacology (2004) 141 1159-1166. There are several others you can find who seem to do what we did - flat whole mount mouse trachea - although they did not describe the pinning down and some can't seem to spell SYLGARD from Dow which it looks like many people use to do this. Hope the pinning explaination was understandable. It is actually easier to do it than explaining how to do it.
-------------- Original message --------------
From: John Kiernan <jkiernan <@t> uwo.ca>
Please tell us how you flattened the curved tracheal cartilages. To me, a tracheal whole-mount is just the posterior (= dorsal) wall of the trachea, which doesn't contain any cartilage.
The tracheal cartilages of a rat cannot be made to lie flat, and for a mouse the exercise must be (forgive the next silly phrase), even more impossible.
----- Original Message -----
From: koellingr <@t> comcast.net
Date: Sunday, July 15, 2007 0:12
Subject: Re: [Histonet] Tracheal Whole Mounts
To: Kim Merriam <kmerriam2003 <@t> yahoo.com>, Histonet <histonet <@t> lists.utsouthwestern.edu>
> Kim, when I did this, I got them flat first (thin pins) and then
> immediately fixed them. Details too lengthy. But if
> you really want to find out about this from someone who is an
> expert, better than me, and that I worked with.... look up
> Thibaut De Smedt in PubMed or Google. He is easy to find
> articles written by him. He was a dendritic cell post-doc
> where I used to work and don't know where he is now but he is
> super nice and can give you all the info you want, far better
> than I on this very subject. Making the mounts and the
> IHC. His work was beautiful.
> Ray Koelling
> Phenopath Labs
> Seattle, WA
> -------------- Original message --------------
> From: Kim Merriam <kmerriam2003 <@t> yahoo.com>
> > Hello everyone,
> > I have been asked to do some IHC staining on mouse tracheal
> whole mounts. I was
> > wondering if anyone has done this; I am looking for
> information on how to
> > flatten out the trachea once it is removed from the animal.
> > Do you think I should fix it first and then flatten it or fix
> and flatten at the
> > same time. Any thoughts or suggestions on how to flatten a
> mouse trachea would
> > be greatly appreciated.
> > Thanks,
> > Kim
> > Kim Merriam, MA, HT(ASCP)
> > Cambridge, MA
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