NetNotes


I have used Lowicryl K4M, LR White and Gold and HM20). I have found HM20 to be superior in: infi ltration, preparation, sectioning and immuno-labelling. I would recommend it over the others, you can make it up in a glass scintillation vial (mixing with light N 2 bubbling). I have also found that acetone is the best solvent for IEM, I have tried both methanol and ethanol-too much extraction. We have done the HPF hybrid technique (light pre-fi xation in phosphate buff ered paraformaldehyde-rinse-HPF in hexadecene-LN 2 storage), followed by AFS. Quickly my protocol is AFS in IEM cocktail (0.2% GA, 0.2% UA in 95% Acetone (5% D-H20) at -90C. T is is followed by gradual increase (slope 5 degrees/hr) to -45C, then held there for solvent rinse (3×10 min pure Acetone) followed by 50% then 75% HM20 in Acetone. Aſt er an overnight in pure HM20, I change to fresh resin once (2 hrs) then again right before UV illumination. Sections are picked up on Formvar-coated nickel or slot grids and immuno-labeled by fl oating on drops of: NH 4 Cl, block, primary antibody overnight 4ºC, TBS rinse, gold-conjugated secondary antibody (2 hrs RT), rinse, hard glutaral- dehyde fi x, rinse, uranyl acetate stain rinse and dry. No fi rst Aby sections followed by secondary Aby serves as negative control. T e key is to make sure the tissue blocks are small and fi t into the planchets, we usually use two 300 μ m.depth planchets fi lled with 1.2 μ l of hexadecene. When sectioning, due to the fact that a good freeze is within a 200 μ m depth of the tissue, we concentrate on the periphery of the tissue, not the middle. Michael Delannoy mdelann1@jhmi.edu T u Oct 1


Specimen Preparation: Unicryl sectioning


We are trying to do post-embed immunolabeling of cell pellets in Unicryl for a client. However, we are having diffi culties sectioning her blocks. T e blocks are extremely hydrophilic; even if the boat is very under-fi lled, water is still attracted to the block face and pulled over the knife. Any sections that we get are pretty much unusable. I have experience sectioning K4M, LR White, and Unicryl before, and the hydrophilicity has never been this bad. T e protocol we used is as follows: Fixation in 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buff er. Rinse in phosphate buff er (3×15 minutes). Dehydration series of 25%, 50%, 75%, 95%, and 3 100% changes with a freshly opened bottle of ethanol. Infi ltration of 1:3, 1:1, 3:1 of 100% ethanol and Unicryl. Ethanol was same freshly opened bottle. 4 changes of 100% Unicryl (2nd one was overnight). Polymerization in Eppendorf tubes in a 55°C oven for 1 day, then a second day once we started having sectioning issues. Some troubleshooting we have done at the microtome includes: Varying the cutting speed and microtome arm cycle speed, varying the thickness setting of the microtome arm advancement, using a new knife and new section of the knife edge, varying the water level from slightly under-fi lled to severely under-fi lled, and varying the block face size and shape. Any advice would be greatly appreciated. Erin Stempinski erin.stempinski@ nih.gov T u Oct 8


I have also used K4M and HM20, one being hydrophilic and later hydrophobic. I have switched to HM20 for the same problems you cite. I would eventually get sections with K4M, but not without a lot of duress. I wonder why you polymerized in an Eppendorf and not BEEM capsules. T e container must be air tight and I know some Eppendorf’s can leak. Also make sure you are adding enough catalyst, by weight not volume as viscous liquids will stick to any pipettes etc. Also I believe the K4M can be polymerized with up to 5% by weight water, so I am curious as to why you are going through 100% ethanol? I can typically stop at 90% ethanol and mix (i.e., LR White) as my fi rst infi ltration step, especially since your protocol does not protect membranes (you can use tannic acid and en bloc uranyl acetate staining for that). If the


54


second day of curing helped your problem, then it’s a polymerization issue. Michael Delannoy mdelann1@jhmi.edu T u Oct 8 Here’s some additional information: Dehydration steps were 15 minutes with 95% ethanol overnight. We dehydrated to 100% for two reasons: 1. We have used this protocol with success previously for this target and 2. Ultrastructural preservation/sectioning was really poor in past experience when doing post-embed immunolabeling with plants and LR White if the sample was not completely dehydrated. We used the Eppendorf tubes because the pellets were very small and delicate (one was just a small smear of cells along the side of the tube) and we were worried about losing them in the transfer to gelatin capsules. T e sheet on Unicryl on EMS seems to indicate that they should polymerize in the Eppendorf tubes just fi ne. Is that not what other people are experiencing? We are currently polymerizing even more by UV at 4ºC. We also got a suggestion to place the samples overnight in Dri-rite which we will also try. Erin Stempinski erin.stempinski@nih. gov T u Oct 8


Specimen Preparation: TEM and SEM on same sample Has anybody done TEM aſt er SEM on the same sample (talking


about soſt animal tissue) with good or acceptable morphology? Yorgos Nikas eikonika@otenet.gr Wed Sep 16


It will depend on tissue and parameters of treatment prior to as


well as fi xation, processing for SEM, observation in SEM and then parameters of reprocessing specs for TEM. It can be done and can be a valuable supplementing info to the images documented by SEM. (will send you an old EMSA-MSA-abstract [1990, with images] with an example of “arterial” SEM to TEM by separate personal mail), Wolfgang Muss w.muss@salk.at Wed Sep 16


Yes. EDS, even. I just had the sections (thin sections) mounted on the TEM grid ready for the TEM, then put them in the SEM. T is works best if the SEM stub has hole drilled in it just less than 3 mm diameter, so that there is a void space below the grid with samples. Be careful though. T e lower kV used in SEM will result in greater beam-stopping by the sample, therefore more energy deposited in the sample and a greater likelihood of rupturing the section or any supporting fi lm. Phil Oshel oshel1pe@cmich.edu Wed Sep 16 I should add that I’ve also done SEM-then-TEM on tissue samples prepared for TEM, en bloc stained or not, then dried and sputter coated for SEM. Aſt er the SEM, “rehydrate” in 100% ethanol, embed and section for TEM. T e morphology in the thin sections likely won’t be as good as tissue processed for TEM, embedded, sectioned, stained (or not), but depending on the study, it can still be useful. Imaging something like (vertebrate liver) bile canaliculi fi rst in the SEM, then in sections in the TEM is a good example of this. Note: the sputter-coated layer of metal causes no issues when sectioning. Phil Oshel oshel1pe@ cmich.edu Wed Sep 16


I did a lot of this in the 1980’s on human ovary. We were looking for particular epithelia cell populations so we would fi nd the cells in the SEM, dissect out the area and then do TEM on that area. As a control we also worked backwards. If we saw the cell population of interest in TEM sections, or semi-thin sections, we would then dissolve out the embedding resin and look at the block in the SEM. Worked quite well and the work was published. It was related to damage done to the epithelia layer of the ovary during surgical procedures. T e ultrastructure in the TEM aſt er SEM preparation wasn’t super good but it gave the information that was required. T is was all pre-PDF and pre-computers so give me a few days and I will dig out my old notes, scan them and send them to you. Allan Mitchell allan.mitchell@ stonebow.otago.ac.nz Wed Sep 16


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