[Histonet] [Fwd: Re: c-fos]

From:Maria Mejia

-------- Original Message --------
Subject: Re: c-fos
Date: Fri, 04 Mar 2005 14:12:05 -0800
From: Maria Mejia 
CC: histonet@lists.utsouthwester.edu
References: <1109952713.422888c9657a4@mail.ucla.edu>

Hello Liao, I would be happy to share my c-FOS protocol & I'm throwing 
in the
ZIF-189 as a bonus. Use (agitiation) in steps from 5 to 15 especially 
the washing

Study of Immediate Early Response Genes using c-FOS & ZIF-189 Antibodies on
Thick Free-Floating Section
1.  Intracardiac perfusion of animal w/saline to wash vascular system 
followed by freshly
made 4% paraformaldehyde/0.1M phosphate buffer (PB) at pH 7.4.
2.  Store brain in 4% paraform. @ 4C - overnight.
3.  Store brain in 30% sucrose/0.1M PB @ 4C - overnight (until brain 
sample sinks to
bottom of container -can add 0.1-0.5% Thermosol (preservative) to 
sucrose solution to
keep for longer period at 4C).
4.  Cut brain using vibratome or sliding microtome section at 40 microns 
thick. Collect
sections in wells containing freshly made 0.1M PB pH 7.4.  

The following is a special long term storing solution for immuno 
sections. Seal container
with sections (well) and store @ 4C for several months:
30gm sucrose
1gm PVP-40 (1% polyvinylpyrolidone, Sigma #PVP40)
30ml ethylene glycol (30%)
Make up the volume to 100ml w/0.1M PB pH 7.4.

5.  Place tissue in soaking solution using gentle agitation @ room 
temperature - 2 hours.
(until bubbles disappear - can use Tween 20 in place of TritionX - no 
8.7 ml Dulbecco's PBS (Sigma #D-5773)
300ul of 10% tritionX-100
1ml of 1% hydrogen peroxide (made from 30% H202)
This solution makes about 10ml
6.  Wash tissue well in 3 changes DPBS - 10-15 minutes each.
7.  Place tissue in modified blocking solution for blocking nonspecific 
binding @ 4C
w/gentle agitiation - overnight.
8.5ml of 2.5% BSA - I used Jackson ImmunoResearch.
300ul 10% Trition X-100 (again can use Tween-20)
1.5ml of super blocking solution (Innovex Bioscience)
100ul normal goat serum

I picked up every third section and did c-FOS & ZIF-SC-189 alternatively:
Before placing primary antibody on sections, leave a bit of the above 
blocking solution
and using a pipette mix in with the antibody solution.

8.  Place c-FOS (AB-5) Oncogene #PC38 Lot #D10535 primary polyclonal 
and Santa Cruz ZIF (c-19) SC-189 Lot #B280 primary polyclonal 
anti-rabbit dilute both
@ 1:10,000 with DPBS use gentle agitation @ room temp - 4 hrs.
9.  Wash tissue well in DPBS X3 changes - 10-15 minutes each.
10. Place in secondary antibody biotinylated goat anti-rabbit (Vector 
BA-1000) dilute
1:1000/DPBS use gentle agitation @ room temp - 30 minutes.
11.  Wash in DPBS X3 changes - 10-15 minutes each.
12.  Place in Vectastain Elite ABC solution & incubate use gentle 
agitation at room
temp - 30 minutes.
13. Wash well in DPBS X3 changes - 10-15 minutes each.
14.  You can use any DAB substrate method with or without metals, e.g., 
nickel or cobalt.
Mix desired DAB solution (agitate) and label - check under scope for 
intensity level.
15. Wash in DPBS X4 changes - 10-15 minutes each.
16. Mount on gelatin-coated glass slides. (I wash and coat my glass 
slides using a home-
made gelatin protocol).
17.  Air-dry slides - 1-2 days.
18.  Dehydrate through x4 changes of absolute alcohol 100% - 10 minutes 
19.  Coverslip in DPX mounting media.

I hope this protocol works as well as it did for me.

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria@ski.org


>hello, I am interested in your protocol for c-fos antibody staining in
>thick free-floating sections. can you share with me your protocol and the
>information about your antibody? Thanks.

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