Problems with Euparal mounts going cloudy and solutions

[billben74]

Hello All,
I had been having problems with Euparal mounts going cloudy. I think I have worked out the problem and certainly I no longer have the problem.
The problem: (Sorry not best image)

DSC00348CloudySlide.jpg (38.95 KiB) Viewed 2301 times

Now look at my set up:

DSC00334FailureSetup.jpg (81.81 KiB) Viewed 2301 times

I had been using those crazy teapots as wash "capsules" to use Walter's parlance.
I had used them with whole mount fructose and had some success.
They are handy as the handle bit is good for pipette resting/forceps resting etc.

I was using warm water fixed onion.


I was also using ethanol not propan-2-ol (I'm a science teacher - I use IAPAC, I mean one of my students might read this
I was also using ethanol to clean my slides.

What I think was going wrong:
a) The onion wasn't being deydrated properly as it wasn't fully submerged during 25%,50%,75%,100% drying.
b) My slides still had ethanol on them (slides wet with etoh) when I put the not properly dried onion skin on them.
c) The ethanol evaporates but tends to leave some water hanging around
--> hence the euparal goes cloudy. Sure Euparal can handle a little water but not much.

Now I didn't just change on thing but hey I wanted nice slides.
Changes:
1) Onion skin now in FAA (my own homebrew made from 40% formalin from aquarium shop 15ml, distilled domestic vinegar 40ml, ethanol (96%) 45 ml)

2) Propan2-ol used for dehydration. I figured I would do as John B did and that perhaps ethanol with its slightly lower boiling point might lead to more water issues. I also used more normal vessels (as in 30 ml wide mouthed glass jars) and made sure the onion was submerged

3) I took more care with drying my slides. I still use ethanol for this but I went to town making sure they were good and dry.

Happily it worked.

DSC00355CNonCloudyHappySlides.jpg (40.18 KiB) Viewed 2301 times

Here are some pics

14_02_201621_49_200021OnionSafarrinONucleoliAndPlasmodesmataSmall.jpg (36.42 KiB) Viewed 2301 times

14_02_201621_49_200021OnionSafarrinOSeveralx400PlasmodesmataSmall.jpg (35.91 KiB) Viewed 2301 times

Would people agree that these are plasmadesmata?

Any comments welcome

[mrsonchus]

Super job Bill, lovely clean mounts. I'd certainly agree re the plasmodesmata - bang-on I'd say.

Fascinating to ponder that the plasmodesmata aren't 'holes' in the plasma-membrane, but may rather be considered 'unpinched' channels where the cell-walls do not actually isolate cells but 'squeeze them into packets' that are then able to exhibit a degree of specialization or even autonomy, but are actually elements of the vast continuum of the plant body, all connected - the plasma-membrane unbroken by these selectively-permeable conduits/channels (desmotubules?)! This fascinates me as it affords several ways of conceptualizing a plant's structure - the "a plant makes cells the cells don't make the plant" position?

Pleased you sorted the Euparal out OK - it's really a case of keeping it simple and consistent with your setup I think - you've yet to get your work-flows established that's all. I went through this stage (and still do with each new technique I try to use) and it soon becomes clear where the inefficiencies and bottlenecks are - many, many times it's a simple case of positioning things where you can reach them once the process begins!

I'd stick to IPA for everything OH if I were you, it's tried, tested and consistent - the azeotrope it forms is easily within the limits of Euparal's tolerance in my experience. Remember to seal your fine mounts after about a week, ordinary nail-polish does a good job - I have all my 'Walter-D' slides safely intact by these methods.

Really nice series Bill, keep up the good work!

p.s. I soon discovered that I needed tissue pieces far smaller than I was using at first, which also makes them easier to process and mount - about 5mm as a largest dimension is a good size you may find. Excellent mounts and crisp colourful images!

[mrsonchus]

Ah, Bill, almost forgot - onion-peels are troublesome in water as you know, they don't like it!
This is my quick & cheap 'vacuum-machine' and it works perfectly for any type of immersion/infiltration - remember to release the vacuum a couple of times then re-apply, helps fluid to flow into the tissue which can collapse as air leaves if you forget this step...

Works very well indeed - very satisfying to watch your tissue sink as the bubbles leave!

ws_vacuum_fixing.jpg (72.37 KiB) Viewed 2291 times

Good luck, keep us informed of your adventures!