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

[Kelly D Mcqueeney]

Hi Maria,

Have you tried c-fos IHC on brain mounted on slides rather than 
processed in a plate?

Thanks,
Kelly

Maria Mejia wrote:

>
>
> -------- Original Message --------
> Subject: Re: c-fos
> Date: Fri, 04 Mar 2005 14:12:05 -0800
> From: Maria Mejia <maria <@t> ski.org>
> To: "LIAO,XIAOYAN" <xyliao <@t> ucla.edu>
> CC: histonet <@t> lists.utsouthwester.edu
> References: <1109952713.422888c9657a4 <@t> 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
> steps.
>
> 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 
> bubbles).
> 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 
> anti-rabbit
> 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.
>
> regards
> Maria Bartola Mejia
> neurohistologist
> Smith-Kettlewell Eye Research Institute
> San Francisco, CA 94115
> Email: maria <@t> ski.org
>
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> 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 
>> the
>> information about your antibody? Thanks.
>>
>>
>>  
>>
>
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