FW: [Histonet] Help with IF in Chamber slide

Lee Crosby lcrosby <@t> echelon-inc.com
Tue Aug 19 17:29:55 CDT 2008

I have always used glass chamber slides. I used NIH-3T3 and 3T3-L1 cells in
DMEM. Each well received 200ul of cells with media. All volumes were 200 ul.
I let them set 2 days and then followed this procedure:

1-Fix cells with 200 ul of paraformaldehyde added to the media (or replace
media with 200 ul of neutral buffered formalin) and incubate for 20 min at
room temp.
2-Wash 3 times with Tris buffered saline (TBS)
3-Permabilize the cells with 0.05% Triton X-100 at RT for 15 min.
4-Wash 3 times with TBS
5-Block with 10% goat serum in TBS 30 min at 37 Celsius
6-Add antibody (anti-PIP3 Echelon Biosciences Z-P345b) and incubate at room
temp for 60 min
7-Wash 3 times in 1% goat serum in TBS
8-Add secondary (goat anti mouse IgM Jackson ImmunoResearch 115-065-075) 30
min at 37 Celsius
9-Wash 3 times in 1% goat serum in TBS
10-Add label (Strept-AlexaFluor 488 Mol Probes S-11223 1:2000) and incubate
30 min at 37 Celsius
11-Rinse thoroughly with distilled water
12-Dry completely
13-Seal with Prolong (Molecular Probes) and store overnight at 4 Celsius
14-Seal edges with your sweetheart's old nail polish
14-View with microscope

We visualized PI(3,4,5)P3 in the cell membranes. So it should work for your
protein as well. If your protein is on the outside of the membrane then you
can eliminate step 3.
Good luck.

Lee Crosby
Echelon Biosciences Inc.
801-588-0455 ext 329

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of karen Cai
Sent: Tuesday, August 19, 2008 10:25 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Help with IF in Chamber slide

I need to do the IF the 8 well Lab-tek chamber slides. Does anybody have
the experience of this? How to culture the cells before blocking? Is
glass type good or using permanox? The cell I used is CHO cell. Membrane
Thanks in advance,

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