NetNotes
Edited by Thomas E. Phillips
Bond Life Science Center, University of Missouri, Columbia, MO 65211
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from April 16, 2009 to June 15, 2009. Complete listings and subscription
information can be obtained at
http://www.microscopy.com. Postings may have been edited to conserve space or for clarity.
Specimen Preparation: leaves something behind on the grid surface. Th is reduced the number
sections falling off grids of sections that I lost. I have also used a very careful quick dip in HNO
3
Every once and a while we run into problems with sections falling off followed by several water washes to make naked grids hydrophilic and
the grids. I know this has been rehashed more than once here, but it’s hard hence make section pick-up from the boat much easier. I had good luck
to fi nd the answers in the archives, so I am asking for the consolidated, with sections sticking where they belonged with this technique. Th e
fi nal analysis of the problem and solutions. I suspect it could be students problem was using the strong acid which dissolved the grid if it was not
using old, oxidized grids, but I’m not sure enough to just give them that washed quickly enough. Patricia Stranen Connelly connellyps@nhlbi.
one answer. I have checked on ‘grid glues’ and searched around, but I
nih.gov Fri Apr 24
knew the experts and experienced would be here. So, what is your You do not need to use strong acid. 1N HCl can do job just fi ne.
strategy? Jonathan Krupp
jkrupp@deltacollege.edu Fri Apr 24 You can keep a grid in this acid for a while without problems. Vladimir
I have had this trouble in the past but not for a long time since Dusevich
dusevichv@umkc.edu Fri Apr 24
I began fl aming the grids. I saw this in the Bozzola and Russell book. For basic counterstaining, a simple acid wash with proper rinsing
Works better for standard grids; be very careful with thin bar grids. Use and drying should be fi ne. I have experienced highly variable section
an alcohol burner with a small fl ame (~1cm max). Pick up a grid with adhesion when I perform immunohistochemistry with any Tween
forceps and sweep briefl y through the tip of the fl ame. It is better to do or Triton detergents, primarily when the grids sink into the reagent
too little than too much since it can be repeated until the desired eff ect droplets (using square pattern, thin bar nickel grids). If this is your
is achieved; aft er a bit you get it right almost always in one or 2 passes. case, then I sympathize. Gregg Sobocinski
greggps@umich.edu Fri
What you are looking for is a “scorched” look, some interference Apr 24
colors in the red and blue range (which must be thicknesses of surface One thing missing from this thread is an explicit discussion of
modifi cation (oxidation?). Th ese grids wet beautifully and sections the technique for picking up sections. I have always picked up sections
cling tenaciously. For the record, I always pick up sections on the shiny from below, and aft er 25 years and thousands of blocks, I have never had
side; I know there are 2 teams on this topic :-) My logic is that it is a problem with sections falling off bare copper grids without adhesive,
like kitchen plastic fi lm that clings better to smooth surfaces. I have either dull or shiny side, with Epon, Spurr’s, or LR White. When you
had wettability issues with gold and gilded grids that can’t be fl amed. bring the grid up from below, there is water between the section and
For these I treat 15 sec in the Harrick Plasma cleaner and they wet the grid, and when the water evaporates, the section becomes bonded
beautifully and sections adhere well. Th is probably works for the tightly to the grid. Th is is not so when you come from above. I should
copper grids as well but I haven’t tried. Dale Callaham dac@research. add that I sonicate the grids in 100% ethanol then place them on fi lter
umass.edu Fri Apr 24 paper in a Petri dish. If you have a problem with the sections running
I routinely use a ‘grid glue’ as I like to immunolabel my sections away when you try to pick up sections, try dipping the grid briefl y in
immersed in the immunoreagents as it makes the whole labeling ethanol, then immediately rinse it thoroughly in distilled water, and
process easier and enhances the labeling, with the antibody/antibodies bring the wet grid to the boat. Th e purpose of this is not to clean the
having access to epitopes on both section surfaces. Th is latter means grid, but to avoid the formation of tiny air bubbles on the grid, which
there is a tendency to lose sections when transferring grids from one tend to repel the sections. You should also rotate the grid as you remove
solution to the next. Anyway my trick is to just briefl y immerse the it from the boat, so that it is vertical when it comes out of the water.
grids in approximately 5ml of chloroform in which about 4-5” of Th is avoids bringing up a large drop of water with the grid, and having
Sellotape has been dissolved (remove the tape itself once the glue has your sections shift when the water is blotted off . Ralph Common
dissolved off it - just shake for a few moments). Th is seems to work for
rcommon@msu.edu Fri Apr 24
me and hope it does for you! Dr. Julian R. Th orpe
bafg3@sussex.ac.uk I, too, was plagued with sections falling off grids. Since I have been
Fri Apr 24 using the following, I have not lost a single section! Dip copper grid
Epon thin sections coming off naked grids during staining into a 0.1N solution of HCl (I usually count 10 seconds with a drop of
procedures was solved when I followed Debby Sherman’s suggestion of HCL on the grid) and blot dry. Dip several times into 100% acetone
putting the grids containing sections into my oven for at least 15 min. to rinse and allow to dry on fi lter paper. I usually prepare the grids
to overnight before staining. I only need to do this occasionally since before I start sectioning so I don’t have to stop each time to get my grids
I do not have trouble frequently (do not know why). I only use my ready. Once my sections are collected, I put the grids into the oven for
oven for this treatment when it is empty because I do not know if the 20 minutes to dry. I can stain immediately aft er removing the sections
fumes that are generated by curing epoxy would have an eff ect on the from the oven or wait until later, it doesn’t seem to matter. Hope this
grids even though they are either in a closed Petri Dish on fi lter paper helps your students! Th ere is nothing worse than spending your day
or in a Grid Box. My newer grids (Electron Microscopy Sciences) do sectioning and seeing only shreds on the TEM! Pat Kysar pekysar@
not need much cleaning, but old ones do. If you sonicate in acetone
ucdavis.edu Fri Apr 24
to remove oil/grease residues, I found it advisable to do a fi nal rinse My technique for cleaning grids is a variation on Pat’s method,
in 100% ethanol before drying since I think the acetone sometimes by using acetone and HCl together. I mix up a 250 ml lot of cleaning
62 doi: 10.1017/S1551929509000418
www.microscopy-today.com • 2009 September
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