[Histonet] embedding cells in parrafin #2

[Helen Fedor]

Making cell tissue blocks

Cells: 
.	Harvest and fix in formalin* for at least 3hrs or overnight (10 fold more formalin)
.	HAVE A NICE VISIBLE SIZE PELLET (1 confluent T75 minimum or 1 T175)
.	Remove formalin* and wash 2x's in cold 1x PBS
.	Cover cells with some 1x PBS and refrigerate

Materials for blocks:
1.	Toothpick or 20ul pipette tip
2.	Agarose  (2% solution agarose with 1xPBS)
ultra pure agarose cat#15510-027 from core

3.	Cells in 1x PBS
4.	0.5ml microtubes (sterile)
5.	Tissue cassettes (1 or 6 compartiment cassettes -Electron Microscopy                                         Science 800-523-5874 cat.# 70078-W)  
6.	New razor blades
7.	Clean cutting board
8.	Container to submerge cassette into with enough 1x PBS to cover cassettes

Method:
.	Suspend cells in 1xPBS - use just enough to pipette cells (1:1 dilution is MAX)
.	Make 2% agarose solution - keep in water bath (42C) so doesn't solidify
.	Make 200ul agarose plug in 0.5ml tube and wait 3 minutes to solidify 
.	Add *120ul of cell solution to .5ml tube and then add *150ul of agarose and pipette up/down ~3 times to mixed well - CUT END OF PIPETTE TIP OFF JUST A LITTLE SO YOU DON'T DESTROY CELL IF NECESSARY
.	Add toothpick and let sit 5 minutes
.	Remove plug with toothpick and cut into sections (if necessary) with new blade on clean surface and add to cassette
.	If you have extra cell plugs then store then in 1x PBS in frig.

* Varies according to how much cell suspension you have. ALWAYS add at least 30ul more of agarose in order for it to make a nice plug.

IMPORTANT NOTES:
.	ALWAYS be sure to label your cassette with an experiment number because that's what the slides will be labeled with.
.	Be SURE to write on BOTH the top of the cassette and down the sides what cell type is in each compartment (if using a 6 compartment cassette)
.	Tell the histologist what you fixed your cells in - if fixed in formalin, ethanol, paraformaldehyde, etc. - this is vital for the processing/embedding process
.	ALWAYS put control cell types in the block  - +/-
.	ALWAYS prepare your block in an asymmetric pattern THIS IS A MUST!! or you will not be able to identify which cell type is placed where on the slide
Helen L. Fedor 

Prostate Tissue Bank, Manager
Oncology Tissue Services, Manager
Johns Hopkins University
600 N. Wolfe St, | Marburg Room 406
Baltimore, MD | 21287-7065

410.614.1660

http://tmalab.jhmi.edu/
http://prostatebiorepository.org/



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Mike Tighe
Sent: Monday, December 02, 2013 12:06 PM
To: histonet <@t> lists.utsouthwestern.edu (histonet <@t> lists.utsouthwestern.edu)
Subject: [Histonet] Embedding cells in Paraffin

Has anyone tried to embed cells grown in tissue culture? I am trying to put some tissue culture cells through same stress as tissue would go through. Fixation, dehydration, and heat. Any ideas? I could re-suspend in OCT and then fix for extended time with NBF but that doesn't quite seem fair.



Thanks for any ideas!
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