Perennial milk-weed - rust-fungus slides

[Hobbyst46]

The method shown is the same as I've seen in old microscopy books...

After a look in the referenced book: Where do you find double razor blades today? I think they are an extinct species, at least as shaving is concerned...

[coominya]

John B. knows a lot more about this subject than I do, but here's a link to look at until he gets back to you. I tried some freehand sectioning many years ago with limited success. With lots of practice one can get a few good ones suitable for making permanent slides.
http://www.ableweb.org/volumes/vol-19/0 ... -YEUNG.HTM

Great link, good primer. When I joined here I had no idea how complex microscopy was, every day I am dumbfounded by the levels of knowledge (and equipment) members here have to pursue their interest. I suppose one day I will find a niche and settle into it but until then I will just have to dabble into all aspects. I Can't help myself.

[desertrat]

The method shown is the same as I've seen in old microscopy books...

After a look in the referenced book: Where do you find double razor blades today? I think they are an extinct species, at least as shaving is concerned...

I think I saw some double edged blades in the local Walmart not too long ago. They're still being made, and certainly available on the web.

If I was going to do this, I would probably buy single edge razor blades. No need to tape one side with them. Those can be found in hardware stores for razor blade scrapers, but aren't as sharp as single edge blades made for shaving, which are still being made by Gem-Personna and available on the web. I bought some to use in an antique Gem single edge safety razor I got on Ebay. It works well, but I eventually got lazy and went back to using an electric razor.

There are also the blades for box cutters, which are bigger and longer. They probably aren't as sharp as shaving razor blades, but it might be possible to hone them. Another project on the back burner which probably won't be worked on anytime soon.

On Ebay there are also new production straight razors, but based on where they're coming from they may need honing and stropping before use.

[mrsonchus]

Two questions if I may. What would you suggest as a beginners plant or plant part to study, and what is an optional method to making the thin sections without a microtome?
Thanks,
Tom

Hi Tom, well for starters I'd suggest a 'soft' stem - leaves are far more difficult to start with. All I use for hand-sectioning is a single-sided razor-blade - a packet of 10 is dirt-cheap!

A Daffodil stem perhaps, although they are a little 'sticky' I suppose, a Coleus is always good as a quite large (diameter) stem is still very easy to section, vegetable stems are always good for the same reason....

I just hold the stem on a bench with one hand and slice-away as thinly as I can with the blade - one single sliding-cut (rather than a 'chop') per section, leave the sections stuck together and separate them after you've cut a dozen or so in some water. There will invariably be some that are good for use - sort them out under a stereo 'scope and stain them then rinse them if you like, or simply use them as they are. All operations are better done with wet tissue and blade.
Here's -> a post I made some time ago <- showing just what may be done very easily by hand...

You'll get some nice sections to peruse, and perhaps try to identify basic tissue-types (by tissue-type I mean groups of cells that all have the same function such as the outer-layer being the epidermis - a tissue of epidermal cells).

Try perhaps to identify these for example,

Xylem
Phloem
Vascular cambium (found between the two above and often 'glistening' or translucent and un-coloured in hand sections)
Epidermis
Parenchyma cells comprising the pith tissue (stem center) and the cortex tissue (beneath the epidermis)
Hairs AKA 'trichomes' - many different types and very interesting in their own right
If you have polarisation you may be able to see crystals in many stem types too!

You'll be amazed just what may be done by hand, and how a nicely-cut section can bring so much pleasure, satisfaction and interest - after your first go you'll very likely be quite hooked!

Cross-wise (transverse) sections of plant ovaries (an ovary may contain just one ovule as with Sonchus - or many ovules as in Daffodils) - e.g. the 'swollen green bit' immediately below a Daffodils' flower - are also very easy to section and the type, position, shape, stage etc of the ovary and the ovules therein will be quite easily seen....

You are about to enter a truly fascinating world with Botanical microscopy - and you don't need to go far to find plants, which can be placed in a small cup of water for several days of study and exploration. Then the urge to compare the same tissue or structures between species may call to you - as it did to me - the rest is history and a very large collection of slides!

Good luck, John B.

p.s. these are -> the ones I use <- £2.50 for 10!

[MikeA]

Hello John,

I am about to embark on a sample collecting mission, armed with chilled aliquots of FAA and hoping to find some new shoots under the fresh blanket of snow that fell last night! Winter is hanging on well past its 'sell by' date here in NE Ohio.

Yet another question - according to the paper 'Fixation, embedding in wax and sectioning protocol' from UNIVERSITY OF LJUBLJANA, VECNA POT 111 BIOTECHNICAL FACULTY their direction is to take sample 'that have at least one dimension smaller than 2 or 3 millimeters.'

That seems rather small to me, but perhaps it is to allow the FAA to more effectively infiltrate the tissue. Also their direction is to keep the samples in a refrigerator at 4°C overnight.

Your thoughts please?

[MikeA]

and yet another...

Where did you find those embedding moulds? I have looked everywhere (even Amazon!) and come up empty. Guess I could get by with something else.

[mrsonchus]

Hello John,

I am about to embark on a sample collecting mission, armed with chilled aliquots of FAA and hoping to find some new shoots under the fresh blanket of snow that fell last night! Winter is hanging on well past its 'sell by' date here in NE Ohio.

Yet another question - according to the paper 'Fixation, embedding in wax and sectioning protocol' from UNIVERSITY OF LJUBLJANA, VECNA POT 111 BIOTECHNICAL FACULTY their direction is to take sample 'that have at least one dimension smaller than 2 or 3 millimeters.'

That seems rather small to me, but perhaps it is to allow the FAA to more effectively infiltrate the tissue. Also their direction is to keep the samples in a refrigerator at 4°C overnight.

Your thoughts please?

I agree, that is unnecessarily small in my experience - I use a pieces about twice that quite often. Let's say you are going to begin with a soft stem - take lengths of about 1cm, then when you have a chance later or the next day, trim them to about 4-5mm long ready for the cassettes and put them back into fixative to finish the 48hr minimum.
As for 4 deg C - I always simply process at room-temp. Factors such as temperature are probably far more important when histochemical analysis and molecular labelling are intended.

The literature can lead you to a very complicated and very time-consuming process, accompanied by the feeling that you can't get the reagents thta 'they' use and so your results will inevitably be inferior - this in my experience is simply not true and I advise you to dismiss such a consideration as it's simply an obstacle to your enjoyment and achievement of your goals.

So, here are a couple of images of some pieces in fixative - if you have any doubts about the size or the permeability of the outer-layers simply make cuts to give access to the reagents. This is how I prepare some larger ovaries, which are an enclosed-unit when collected. I simply shave a thin slice from some of the edges that are not an important part of the proposed sections, such as the outer-case of the ovary, to aid reagent entry, as you may be able to see with these 'squared-off' ovaries in the second image below...

ws_small_snowdrop_fixative (1).jpg (41.86 KiB) Viewed 13469 times

and,

ws_small_snowdrop_fixative (11).jpg (25.12 KiB) Viewed 13469 times

I have a very large amount of info here in this forum in my posts of all sorts of tissue preparation and slide-making that you'll find really helpful also. If you select the 'print view' this forum provides, the right-click and 'print to PDF' with Windows, you can archive my posts for later reference also - that's what I do!

Hope this help you to get started. Are you intending to try to make wax-blocks, sections then permanent slides?

John B.

[MikeA]

Hello John,

Thanks for getting back to me and confirming I do not need to take such small samples - I admit my tendency in the past was to go the other way and take HUGE samples that were way too big when it came to trying to do thin sections.

Had to laugh just a bit at your first photo that includes the inevitable 'cuppa - you would have caused a riot in some of the labs I was associated with!

When I lived in England, we had a lab in Derbyshire and having spent most of my working career around labs here in the states I was 'gobsmacked' my first trip to see the more relaxed attitude - frankly I thought it was more a manifestation of allowing responsible adults act as they normally would! We Yanks have a tendency to try to legislate responsible behaviour, something that will never work as idiots will be idiots.

Responding to your points out of order, yes, I would very much like to embed the samples in wax blocks, cut the thin sections, then go through the dehydration (I need more info from you on how you do that!) and then stain and make permanent mounts. I have all the supplies (at least I think I do) and equipment - two rotary microtomes, a disposable blade holder (TissueTek) as well as a 'new' AO microtome blade; Coplin jars and all kinds of other 'necessary' things - now it is just a matter of doing something with it.

The wax moulds shown in your photo appear to be a commercial item but I have not been able to track anything down as of yet.

I will be doing some printouts of your various posts - there is a wealth of information there that I expect you earned the hard way, and I appreciate your willingness to share. Thank you! I also have a PID controlled oven and a refrigerator at my disposal; I really cannot fathom why that procedure from the University of Ljubljana insists on the FAA being cooled to 4°C or why they would leave the samples in the refrigerator overnight; truth be known it seems to be contraindicated, but what do I know?

Inevitably I will be back knocking on your door for some more information as I blunder through the various steps.

[mrsonchus]

Aha! You're most welcome old chap - always good to see another having a run-at slide making! You simply won't believe how interesting the entire process is, let alone the plant anatomy and it's attendant taxonomy!

Here are a few links to some of my posts that you may find interesting,
-> root-tip mitosis <-

-> liverwort - a whole organism <-

-> complete protocol - the early work.. <-

Here's a snip of one of my posts re the entire process and it's tendency to seem very much more complex in the literature than I've actually found it to be,

start snip -------------------------
Take a step back from Ruzin's really very long (in terms of stage duration) protocols to realise that most herbaceous tissue needs far less time at each stage than the 4hrs+ often written by Ruzin.

Start with a soft stem (stems are so much easier to orient when embedding in wax) tissue, say Daffodil flower-stem.

Stages in a nutshell,
kill it (FAA-50 for >=24hrs)
dehydrate it (Isopropanol series)
remove alcohol with wax-antemedium or 'clearing agent' - (Histoclear)
infiltrate with molten wax
embed in wax
section from wax-block
de-wax with wax solvent (Histoclear)
remove Histoclear with alcohol
remove alcohol with water
stain with aqueous stain such as Safranin
dehydrate with alcohol
stain with alcohol-base stain such as Fast-green
remove alcohol with Histoclear (Histoclear is compatible with resinous mountant such as 'Numount' of 'Omnimount')
mount in resinous mountant

Hope that helps a little -
Ruzin can make the process seem quite gargantuan - it's not easy, but it's not as difficult as Ruzin's book may suggest.

end snip-----------------------------

Here's the link to the post.

John B.

[MikeA]

Hello John,

Progressing but ever so slowly - I have printed out those specific posts and lots of information but also have run smack into a dilemma - Histoclear! Not sure what the supply situation is in the UK for this material, but not to put too fine a point on it - here in the USA it sucks! We are looking at pricing of approximately at least $89/gal (and that is the small US gallon, not the big Imperial one!) up to $257/gallon.

Needless to say I think I will be just going with the old standard Xylene - is there any significant improvement in functionality with the Histoclear other than the elimination of the vapor/odor? I am old anyway so not too worried about what breathing it might do to me.

I can get Xylene/Xylol from the local DIY for <$20/gal. For that difference I will just hold my breath a bit longer!

We used to wash our hands in benzene, trichlor, heptane, petrol, you name it, so the damage is already done in all likelihood.

[mrsonchus]

Hello John,

Progressing but ever so slowly - I have printed out those specific posts and lots of information but also have run smack into a dilemma - Histoclear! Not sure what the supply situation is in the UK for this material, but not to put too fine a point on it - here in the USA it sucks! We are looking at pricing of approximately at least $89/gal (and that is the small US gallon, not the big Imperial one!) up to $257/gallon.

Needless to say I think I will be just going with the old standard Xylene - is there any significant improvement in functionality with the Histoclear other than the elimination of the vapor/odor? I am old anyway so not too worried about what breathing it might do to me.

I can get Xylene/Xylol from the local DIY for <$20/gal. For that difference I will just hold my breath a bit longer!

We used to wash our hands in benzene, trichlor, heptane, petrol, you name it, so the damage is already done in all likelihood.

Hi Mike, yes I pay about £120 per US Gallon for it from 'Geneflow' over here! There is also a Histoclear 2 and a Histoclear 3 - both of which reduce the smell or orange-zest substantially - not that it has ever bothered me in the slightest - it's not even that strong if you don't throw the stuff around the lab!

Both version 2 & 3 are significantly cheaper than Histoclear 1 - and I've still got some of both left - they're hopeless when it comes to mounting with resinous mountant - I quickly went back to Histoclear 1....

That said, as I gained experience I found my rate of usage of Histoclear dropped so dramatically that it soon became no real problem to buy a US Gallon about once a year - and I make a lot of slides!

I know what you mean re Xylene - the warnings are probably most relevant if you use it every day for 50 years... But I use Histoclear as it is not only non-toxic but easily disposed of.

I also had the advantage when I started of being able to buy it by the 250ml bottle from the superb U.K. 'Brunel Microscopes' - from whom I regularly bought all my supplies when I started, including a new Trinocular compound and a stereo-zoom binocular 'scope.

I'd say go ahead and use Xylene to begin with - you can always change to Histoclear if you begin to make large numbers of slides and begin to worry about over-exposure.

All the clearing-agent (be it Histoclear or Xylene) has to do is to replace the dehydrating alcohol and from there the tissue is to be placed into infiltrating wax, and to dissolve the wax from your sections that have been dried onto slides pre staining & mounting.

The fact is, as I can say for sure due to the recent trials I have made slides from, that a clearing agent isn't really needed to remove the alcohol - the tissue, fully dehydrated, may be moved straight into molten wax without any problems.

You will however still need to use a wax solvent to de-wax the slides.

I'd carry-on with your proposed plan if I were you my friend - you should be absolutely fine with Xylene.

Study the posts of mine that include complete protocols - I nearly always post the protocol/s used for my posts straight from the whiteboard 'protocol-boards' that I use on my lab wall during live runs.

Remember you really won't need a protocol that has 10 dehydration stages and 5 infiltration stages taking 5 days - not needed at all!

Here are a couple of examples, with and without Histoclear clearing,

ws_protocol histoclear vs alcohol comparison 07_02_2018.jpg (82.95 KiB) Viewed 13442 times

The results for comparison,
First two images of the alcohol-only protocol,

and here the alcohol-only has preserved the sieve-plate perfectly during sectioning - not too soft/hard...


A couple processed in the 'normal way' including the use of Histoclear for clearing and wax ante-medium,

and,


You can see no real difference at all, and Sonchus are in my experience troublesome blighters to section - very delicate collenchyma.

and, my '10-hour extreme' protocol... I started 'by the books' at about 90 hours, eventually perfecting the 10-hour version.

ws_10_hour_extreme_protocol (1).jpg (77.52 KiB) Viewed 13442 times

ws_10_hour_extreme_protocol.jpg (138.51 KiB) Viewed 13442 times

Hope this helps a little - good hunting!

John B.

[desertrat]

I use hardware store Xylene as a clearing agent for zoological slides. These are whole mounts of tiny pond critters and insect innards. It works fine, but is more sensitive to traces of water in the last alcohol than some of the commercial clearing agents. The last alcohol needs to be nearly absolute. I've discovered that Kleanstrip SLX denatured alcohol is around 98.5 % on my alcohol hydrometer, and mixes with hardware store xylene without making the mixture cloudy. In theory, 1.5% water should be too much and should make the mixture cloudy, but I think some of the other ingredients/contaminants somehow prevent the cloudiness from forming.

However the water content in hardware store denatured alcohol isn't carefully controlled, so you need to test the alcohol and xylene for miscibility before proceeding. SLX denatured alcohol is a roughly 50/50 mixture of ethanol and methanol with traces of a ketone or two and maybe a little ethyl acetate. It's the most easily obtained denatured alcohol in my local area. The local Walmart sometimes has it.

Speaking of hardware store xylene, it contains a mixture of xylene isomers and 10% to 30% ethyl benzene. But the physical properties of ethyl benzene are almost identical to xylene.

Some of my slides made with these products are 8 years old and still look fine.

[Hobbyst46]

Hi
@MikeA
@desertrat
I only read what you say about these liquids. Later I will read JohnB's. But:
1. Age is not an excuse to dip one's hands in suspected carcinogens and sniff their vapors in between.
Benzene is a proven established carcinogen. Ethyl benzene - same as benzene as I recall. Toluene - not yet. Xylene - suspect. I would avoid them all.
2. There is no known exposure threshold of cancinogenicity - AFAIK.
3. If one must use xylene, or ethyl benzene, or toluene, he/she should wear appropriate gloves and protect their breath with appropriate equipment, not "hold it" IMO.
4. The problem with cancer is not only death, but the terrible suffering and pain that precedes it. Science has no answer to this as yet.(*) excuse me for ruining the party...
Best regards

[MichaelG.]

[ ... ] Hope this helps a little

Thanks for posting that excellent summary, John
... an invaluable reference ...

MichaelG.

[MikeA]

Hello JohnB,

Again, do not know how to thank you for sharing your hard-earned information; I have no doubt it will short cut the rather steep learning curve dramatically and help me expand my enjoyment of this hobby!

About the only downside is my having to order more ink cartridges for my printer - it has been working overtime printing your your posts!

We are still in the ghastly, ghoulish grasp of winter here in NE OH - current temp is 41°F, so not quite 'spring like.'
As a consequence all the hoped for young shoots are refusing to poke thru the soil (to be summarily decapitated by me! ) At least it gives me more time to assemble the necessaries.

Still looking for a source for the wax moulds - can you direct me on that?

[MikeA]

Hello Hobbyst46,

I certainly respect your positions.

[MikeA]

Hello John,

Progressing but ever so slowly - I have printed out those specific posts and lots of information but also have run smack into a dilemma - Histoclear! Not sure what the supply situation is in the UK for this material, but not to put too fine a point on it - here in the USA it sucks! We are looking at pricing of approximately at least $89/gal (and that is the small US gallon, not the big Imperial one!) up to $257/gallon.

Needless to say I think I will be just going with the old standard Xylene - is there any significant improvement in functionality with the Histoclear other than the elimination of the vapor/odor? I am old anyway so not too worried about what breathing it might do to me.

I can get Xylene/Xylol from the local DIY for <$20/gal. For that difference I will just hold my breath a bit longer!

We used to wash our hands in benzene, trichlor, heptane, petrol, you name it, so the damage is already done in all likelihood.

Hello JohnB,

Again, do not know how to thank you for sharing your hard-earned information; I have no doubt it will short cut the rather steep learning curve dramatically and help me expand my enjoyment of this hobby!

About the only downside is my having to order more ink cartridges for my printer - it has been working overtime printing your your posts!

We are still in the ghastly, ghoulish grasp of winter here in NE OH - current temp is 41°F, so not quite 'spring like.'
As a consequence all the hoped for young shoots are refusing to poke thru the soil (to be summarily decapitated by me! ) At least it gives me more time to assemble the necessaries.

Still looking for a source for the wax moulds - can you direct me on that?

[mrsonchus]

Hello JohnB,

Again, do not know how to thank you for sharing your hard-earned information; I have no doubt it will short cut the rather steep learning curve dramatically and help me expand my enjoyment of this hobby!

About the only downside is my having to order more ink cartridges for my printer - it has been working overtime printing your your posts!

We are still in the ghastly, ghoulish grasp of winter here in NE OH - current temp is 41°F, so not quite 'spring like.'
As a consequence all the hoped for young shoots are refusing to poke thru the soil (to be summarily decapitated by me! ) At least it gives me more time to assemble the necessaries.

Still looking for a source for the wax moulds - can you direct me on that?

Hi, you're very welcome. Ah - the steel moulds are easy to find - these are the ones I buy - take my advice - buy the medium-sized first and a couple of the biggest deepest ones - they're handy for holding tissue-pieces in molten wax during embedding.

Here's -> the link to the supplier...

If funds allow start with about a dozen. Sorry I'm short of time now, but tonight I'm on-line from about 10pm as usual.

An image from the listing,

s-l500.jpg (10.51 KiB) Viewed 13420 times

I'd at least start with a couple of each maybe not the tiniest one yet. If the listing says he has none - just try the search slightly differently of check his other items as the listings can be multiple and a little mixed-up I find....

In fact, the image and link above is for his Histotech ones, he also has Shandon ones for sale in another listing - they're both the 'real-deal' pro-lab versions...
He's always a reliable source - I buy from him quite often.

regards, John B.

[MikeA]

Hello John,

Thanks for the quick response.

Afraid your local supplier has some rather expensive shipping costs! For 6 of the few sizes he has in stock, the item cost is $14.62 but the shipping is >$47 - Yikes

He may be the 'only game in town' and seems to be doing well with his business - I understand they are all used so he probably is getting them for next to nothing, so good for him.

Obviously I need to come up with an alternate plan - thinking of making the molds out of PTFE - I have some raw stock as well as milling machines.

There is also listing for disposable vinyl plastic cryomolds, in various sizes, case of 100 so might just go with that to get started.

[mrsonchus]

Hello John,

Thanks for the quick response.

Afraid your local supplier has some rather expensive shipping costs! For 6 of the few sizes he has in stock, the item cost is $14.62 but the shipping is >$47 - Yikes

He may be the 'only game in town' and seems to be doing well with his business - I understand they are all used so he probably is getting them for next to nothing, so good for him.

Obviously I need to come up with an alternate plan - thinking of making the molds out of PTFE - I have some raw stock as well as milling machines.

There is also listing for disposable vinyl plastic cryomolds, in various sizes, case of 100 so might just go with that to get started.

Good grief! That's a bit steep for postage! What type of clamp/s do your 'tomes have? To hold these cassettes they need the right type of spring-loaded clamp rather than the screw-down clamp that will hold all manner of objects such as wood-blocks with wax-blocks mounted upon.....

ws_sledge microtome arrival day (1).jpg (199.54 KiB) Viewed 13409 times

You can still use the cassettes for processing, and I have found it's perfectly possible to mount wax-blocks cast from silicone ice-cube trays onto the back of a cassette with a little trimming and a hot spatula. Or just use wooden blocks to mount the wax-blocks - see my youtube video,


The exact-same method may be used with the back of the cassette in place of the wood-block in the video - depends what type of clamp you have on your 'tome....

Some other moulds I've used as well as the steel ones,

ws_small_casting spare wax (2).jpg (28.9 KiB) Viewed 13409 times

John B.

[MikeA]

Hello John,

Yes, I thought it a bit pricey but guess it is 'supply and demand'!

Here are some photos of the specimen holders in both of the microtomes - as you can see there is the remains of one of my very early attempts from perhaps 12 years or more ago using my venerable AO820. Both it and the Leitz have the clamp type holder with one I believe being able to take either the moulds or something else by use/removal of the Al block/backer.







Think I will start out with making a mold or finding something that will suffice and going with the wood block (as I recall it actually worked reasonably well!) I have no desire to do any commercial preps so my volume will be modest.

Again, I cannot thank you enough for your help and generosity with all your knowledge.

[mrsonchus]

Sounds good - the wood-block mounted onto wax-block combination works as well as the cassette does - this I can tell you from experience - no need to worry that you will not be getting the same quality as with cassette-backed wax-blocks, you will. The major advantage of the cassette system in respect to the sectioning stage is the ability to place the wax-block in exactly the same position each time.
When sectioning a dozen different blocks in one session this is a very, very big help. Also of course being able to go back to a single block - without the need to leave it in the chuck 'til you finish sectioning it.

I make certain that the orientation of the specimen in wax (i.e. during the embedding stage) is exactly right for each block - this avoids the need to alter the angle/s of the chuck during sectioning, thus achieving the above-mentioned consistency. This can be fine-tuned with trimming of the wax-block prior to mounting upon the wood-blocks - see my short video where I do exactly that.
With the cassette system you don't get this option - a 'wonky' (i.e. non-perpendicular to the sectioning-plane) stem embedded will require alteration of the chuck's angles - thus losing position for other cassettes....

I would recommend the blue silicone ice-trays or the pyramid 'waffle moulds' for smaller pieces to start with. They are easily removed when set and easily mounted onto in your case wooden blocks.

Good luck, let us know how you get on!

[MikeA]

Hello John B,

With my earlier, rather Heath Robinson'ish efforts were surprisingly workable, even considering I used some paraffin from candles that had accumulated from who-knows-where and the fixation/infiltration/etc./etc. was catch-as-catch-can, the results were not horrible!

With your guidance and a bit more of a studied approach I am optimistic I can make presentable slides. Towards that end I 'captured' some raw material just a bit ago - an onion that had decided to sprout, presenting a perfect starting point, and into the fixative it went! Still a bit curious about why several sources mentioned keeping the FAA refrigerated, very specifically at 4°C, and keeping the samples in the fixative for 24 hours in the refrigerator - I wonder if it is to reduce tissue stress?

At any rate, proceeding apace with my mrsonchus manual by me side (and yes, the new ink cartridges for the printer came today. ) so all printed out, punched, and ensconced in a dedicated looseleaf notebook.

Lots of fun ahead and I expect some long hours of working to learn this craft, but enjoyable time.

[mrsonchus]

Hello John B,

..............

Lots of fun ahead and I expect some long hours of working to learn this craft, but enjoyable time.

You're in for a great time my friend - just let me know if you have any problem or questions - chances are high that I've had the same ones!

John B.

[MikeA]

Hello John,

Just a couple of thoughts - perhaps the reason those other sites recommended the refrigerated FAA is to lower the tissue damage when the sample is first cut - just a guess on my part, but cannot think of anything else.

Also on the mounting blocks - think I will just machine some from UHDPE and mill the crosshatch pattern on the face - what do you think? I can make them to fit the holders on the microtomes and be off to the races.

Ordered what I hope is the correct ice cube tray - our refrigerators have the built in ice cube makers, so have not done anything with trays in many years! Went with one that makes cubes 0.8" (~20mm) on a side, so figure I can pare off any excess wax.

So much to learn, so little time....

[mrsonchus]

Hello John,

Just a couple of thoughts - perhaps the reason those other sites recommended the refrigerated FAA is to lower the tissue damage when the sample is first cut - just a guess on my part, but cannot think of anything else.

Also on the mounting blocks - think I will just machine some from UHDPE and mill the crosshatch pattern on the face - what do you think? I can make them to fit the holders on the microtomes and be off to the races.

Ordered what I hope is the correct ice cube tray - our refrigerators have the built in ice cube makers, so have not done anything with trays in many years! Went with one that makes cubes 0.8" (~20mm) on a side, so figure I can pare off any excess wax.

So much to learn, so little time....

Hi mike, hmmm - not sure about UHDPE - remember you need a hot spatula for the wax-block-to-block mount process.... Personally I'd just use a nice piece of hardwood - just make-up half a dozen that are exactly the same size. Wood resists just about everything and you really do (quite surprised me at first I must admit) get a very strong wax-wood bond with the simple method shown in my video - no need for any type of keying sculpturing (e.g. grooves in the wood) needed.

It took me about half an hour to make several from an old wood-angle cutting guide. Drop them into molten wax, take them out then just scrape as much wax off as you can for starters - then you'll find they are ready to use....

I think the use of lower temperature fixative may well be related to the use of immunohistochemistry and sensitive molecular markers.

Sometimes, such as when trying to stop mitotic chromosome travel along spindles in 'it's tracks' as it were, to enable nice mitotic images/figures for microscopy, a faster fixation is desirable - more of a room-temperature scenario I think.

Why not trial both?!

Great fun ahead for you my friend!

John B.

[MikeA]

Hello John,

As usual, good thoughts re the hardwood and simple enough to make - pretty sure I have some red oak in the garage and it would be an ideal candidate.

Found a source for those moulds, or at least maybe found a source - https://www.ebay.com/itm/Tissue-Embeddi ... 1438.l2649

That seller lists a number of sizes and shipping is free, albeit slow.

Thinking of buying a selection, per your earlier post, including sizes (all in mm):

15X15X7
24X24X7
30X24X7
60X37X10 or 60X37X13

What do you think?

[mrsonchus]

Hello John,

As usual, good thoughts re the hardwood and simple enough to make - pretty sure I have some red oak in the garage and it would be an ideal candidate.

Found a source for those moulds, or at least maybe found a source - https://www.ebay.com/itm/Tissue-Embeddi ... 1438.l2649

That seller lists a number of sizes and shipping is free, albeit slow.

Thinking of buying a selection, per your earlier post, including sizes (all in mm):

15X15X7
24X24X7
30X24X7
60X37X10 or 60X37X13

What do you think?

Sounds good to me my friend - go get 'em!

John B.

[coominya]

Hi Tom, well for starters I'd suggest a 'soft' stem - leaves are far more difficult to start with. All I use for hand-sectioning is a single-sided razor-blade - a packet of 10 is dirt-cheap!

Well the blades arrived today, so tomorrow I'll go find some stems and try my hand at sectioning. After I've done a bit of observing I'll check back and pick your brain for a simple preservative/stain formula. I won't be keeping them forever "yet" but it would be nice to have some samples in a jar for a few weeks like I do with the protista. Little steps for baby feet eh.

[mrsonchus]

Dead-right my friend. We all have to start at the beginning. You'll find your technique will improve very quickly indeed - remember to keep the sample and the blade wet and 'slice' rather than chop if you can.
You'll be amazed what can be achieved with basic hand-sectioning!

Good luck, let us know how you get on.

John B.

p.s. Have a peek at JimT's posts for hand-sections - he's rather good at them!