RE: Immunostaining cells on Matrigel -- how we do it

From:Philip Oshel

If I may, there is a cheap and easy way to make your own plastic 
culture dish with an inserted coverslip for use with an inverted 
First, find a cork-borer smaller than the coverslips you what to use. 
For 12 mm rounds, I use a #4 borer. From our borer set -- I don't 
know how standard these numbers are.
Second, get a cork-borer sharper. That odd cone-shaped thing with the 
swinging flap of metal that no one knows what it's for. Sharpen the 
borer well.
Third, get a bunsen burner. (Yes, I know, buy hey, I'm an Old Fart 
who's used these since junior high without killing myself or burning 
down any buildings.) A propance torch also works.
Turn on the burner, get a nice inner blue cone in the flame, and hold 
the cutting end of the borer in the flame. Cutting end up -- borers 
make nice chimneys, and if the cutting end is down, the hot gases 
will go up to your hand. Get the end nice and hot, but it *doesn't* 
have to be glowing.
Press into the center of the culture dish. You will quickly cut a 
nice, clean, round hole.
Surround the hole with your favorite cell-satisfying sticky stuff. 
Non-toxic silicone sealant, fingernail polish, whatever.
Apply coverslip and let dry thoroughly. Check for completeness of 
seal. Several can be made in relatively little time.
We use these for cells in culture and zebrafish embryos, and both seem happy.
Or as happy as they can be after being poked and prodded and labeled and ...

Mind, I've also used the chamber slides and commercial culture 
dishes-with-coverslips and been pleased.


>Charlene and interested others:
>Immunofluorescent staining can be accomplished on 24 well plates, if 
>you have an inverted fluorescent or confocal microscope with which 
>to view the stained plates. Sometimes the availability of the 
>appropriate end product observation tool limits what we do in the 
>lab for our investigators. Hence the proliferation of many useful 
>product to grow and observe cultured cells. Below are a few we have 
>used in our lab.
>Chamber slides: Available from Nunc, Fisher Scientific and probably 
>others. These slides come in single well up to 16 well formats with 
>various options for growth surface treatment, e.g. matrigel, 
>fibronectin, collagen type I etc. These slides have a gasket and 
>plastic well that contains the cell media for growth. After cells 
>have been grown on the slides, we fix with 10 % NBF and stain using 
>standard immunofluorescent techniques. Remove the chamber wells and 
>gasket, apply fluorescent mounting media e.g. vectashield and No 1 
>cover slip of sufficient size. Note: we use no 1 (thickness) cover 
>glass for all fluorescent applications because the resultant images 
>are sharper.
>BioCoat inserts: We get ours from Fisher. These cylindrical inserts 
>have a porous membrane on the bottom impregnated with matrigel, 
>fibronectin, collagen type I, etc. They fit into standard multiwell 
>tissue culture plates. Cells grown on these inserts may adhere to 
>the membrane or pass through it to the plate below. Many 
>investigators use these to assess the invasive capacity of tumor 
>cells in vitro. The good news is that these inserts can be, fixed 
>and stained in place or on another plate according the investigative 
>question. The membrane can be cut out using a Number 11 scalpel 
>blade or exacto knife floated onto a standard microscope slide, 
>cover slipped, mounted and viewed on a standard scope.
>Of course, if you have access to an inverted fluorescent or confocal 
>scope, you can fix and stain your cultured cells in the plate and 
>view them directly simplifying matters greatly.
>Hope this helps, Donna Montague, Center for Orthopaedic Research, 
>UAMS, Little Rock, AR
>-----Original Message-----
>From: Carlene L. Zindl []
>Sent: Tuesday, November 26, 2002 4:16 PM
>Subject: Immunostaining cells on Matrigel
>Hi all,
>Does anyone have a protocol or good reference for performing 
>immunofluorescent staining on cells grown on Matrigel?  Should the 
>cells be grown on coverslips or is the staining possible in 24 well 
>plates? Thank you for any info and I hope everyone has a happy 
>Washington University
>St. Louis, MO

Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)

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