Re: [Histonet] [Fwd: Re: c-fos]
Have you tried c-fos IHC on brain mounted on slides rather than
processed in a plate?
Maria Mejia wrote:
> -------- Original Message --------
> Subject: Re: c-fos
> Date: Fri, 04 Mar 2005 14:12:05 -0800
> From: Maria Mejia
> To: "LIAO,XIAOYAN"
> CC: firstname.lastname@example.org
> References: <email@example.com>
> 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 each.
> 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: firstname.lastname@example.org
> LIAO,XIAOYAN wrote:
>> 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
>> information about your antibody? Thanks.
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