50, 75, 95, and 100%). Tissues were washed in anhydrous etha- nol and infiltrated in 50% HM20 resin with Z6040 embedding primer, followed by 100% resin exchanges with Z6040 (leſt over- night with 4 exchanges every hour or so). I then cured the tissue at -20°C with UV light with fresh HM20 resin with Z6040 for 24 hours. HM20: mixed for 2 hours with N(g) bubbling. I also used a microwave processor during the processing. Trial 2 - I did a long graded ethanol dehydration series (25, 50, 55, 65, 75, 80, 85, 90, 95, and 100%) followed by anhydrous eth- anol washes. I also increase the HM20 resin exchanges to three days (2 overnights with a total of 6 resin exchanges). Every- thing else is the same as trial 1. Trial 3 (in process) – Using high pressure frozen (HPF) tissue in 1-hexadecene as the cryo-protectant HPF samples were placed in an immunoelectron microscopy cocktail (0.5% glutaralde- hyde, 0.1% UA in acetone) and leſt in a liquid nitrogen dewar until free substitution. Rudy Alvarado

Tanks for the detailed protocol. Te second protocol looks

good; I can see problems with the 1st protocol. If the 3rd proto- col does not work, I suggest starting infiltration with 20% HM20 resin and gradually moving to 100% with 2 days of time at 4°C to preserve immunogenicity. Also use a control of pure resin with- out any tissue sample to check performance of the resin. Some- times the resin absorbs moisture if the bottle is leſt open and this makes polymerization difficult. Regan M. We’ve discussed this on several occasions, and it is said

(by Heinz Schwartz among others) that the HM20 monostep can be difficult due to partial polymerization prior to use and that some brand new bottles in fact are not OK. Randi Olsen

I think bad polymerization may be the issue. It says in

Electron Microscopy Science’s HM20 technical data sheet that initiation of polymerization is largely independent of tempera- ture, blocks may be polymerized at the same temperatures which are used for infiltration. I wonder if you infiltrated your samples at -20°C for the last few steps before curing them at -20°C. Another thing may be the embedding molds. I usually use BEEM and sometimes gelatin capsules. I have experienced bad polymerization with flat embedding molds. Gang (Greg) Ning

We purchase the Lowicryl HM20 kit from EMS. As for

infiltration I do exchanges with a microwave (3 mins @ 120 W with 20 mm Hg vacuum pressure) followed by 30–60 minutes on the orbital rocker at 4°C. On the last infiltration exchange I let it sit at -20°C for 60–120 minutes before UV curing it at -20°C in the free substitution unit. I think it might be the flat BEEM capsules we are using. On the HPF/FS samples I will try both flat BEEM capsules and gelatin capsules. Z-6040 embed- ding primer (from EMS) is supposed to help with sample and resin adhesion, and to prevent the sample from pulling away from the resin when sectioning. I don’t know the chemistry behind this. Lastly, I have never had bad effects from using it. Rudy Alvarado

I’m a little astonished with how much voodoo comes up

in this HM20 thread. HM20 is really not complicated to use. 1. Make sure you don’t bubble air through the resin. Mixing the components carefully by pipetting without bubbling is enough. It’s stable in the fridge/freezer for at least a month. 2. Polymerize the resin under nitrogen gas (=in the AFS) if you have open/leaky

2020 January •

vials. 3. If you can’t polymerize under nitrogen, make sure your vials are filled to the top with resin and seal them as well as you can. 4. Polymerize 24–48 h under UV, if it doesn’t polymerize by then it will never polymerize (Te only thing that will happen is that the unpolymerized resin will slowly evaporate). Let the sample sit open, overnight in the hood for any unpolymerized resin to evaporate. Tis should give you dry, hard blocks. Tey can be soſt at the top if they were in contact with air. If you have infiltration problems (which is most likely the

case based on your description), do more and longer dehydra- tion/infiltration steps. Tere is a slight viscosity difference between the solvent and HM20, which in dense tissues can cause poor infiltration. If you start infiltration with 25% HM20 in solvent that problem should go away. Also keep in mind that 1-hexadecene has a melting point

of 4°C, it will be solid in the freeze substitution. If your sample is surrounded by it, the excess solvent in the FS will dissolve it. If it’s deep in your tissue (= you threw the tissue into hexadec- ene and let it sit) it’s not going to come out at -20°C. Z-6040 is a silane (dive into the MSDS [3-(2,3-epoxy-

propoxy)propyl]trimethoxysilane). I struggle to understand why you would want to use that, particularly for HM20. Te common use is for materials science samples where you have a flat surface of some solid material that doesn’t bond with the resin, such as metal foils. Te silane covers the inert surface and has an epoxy group that can now crosslink with an epoxy resin mixture. I’m also not a fan of applying a vacuum to resins because you’re not entirely sure what resin components evapo- rate and what stays behind. Chris Buser

Folds in sections of cells grown on

Transwell filters I am doing TEM of embryonic cells grown on polycarbonate

membranes in Transwell dishes. Te cells grow in layers of two or three cells. Aſter fixation (aldehydes, osmium) I remove the filters with the cells on top, cut them into little pieces, and proceed to dehydration and embedding in Eponate 12 placing the pieces on flat molds. I cut (ultra-thin, 60 nm) the samples perpendicular to the membrane. I get lots of folds and wrinkles that radiate from the membrane to the cells…a pity, the cells look really nice but it is hard to image because of the many folds. Has anyone done TEM of cells in Transwell or other types of filters and have any suggestion on how to avoid the folds? Tank you!! Amalia Pasolli

Te cause of the folds may be the thinness of your sections.

Try cutting at 80 nm or if you need your sections to be so thin you can try grids with formvar or carbon. Juan Carlos Leon

Recently I’ve experienced the same problem with chemi-

cally fixed cells on Transwell (PES) filters and on track-etched polycarbonate membranes, dehydrated in ethanol and embed- ded in EPON (Mollenhauer based mixture, medium-hard). In my case sectioning thicker doesn’t help that much, the wrin- kles are already visible when the sections come off of the dia- mond knife. Only sections less than 40 nm thick resulted in less wrinkling. Additional stretching with chloroform vapor doesn’t help. I haven’t tried stretching with a heat pen or add- ing a drop of 100% ethanol to the water bath (I avoid the lat- ter since it makes it almost impossible to section ribbons). Te whole behavior of the material during sectioning appears to be due to a difference in sectioning properties between the


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