-----Original Message-----
From: Maria Mejia <maria@mail.ski.org>
To: histonet@pathology.swmed.edu <histonet@pathology.swmed.edu>
Date: Monday, September 11, 2000 7:38 PM
Subject: c-Fos antibody protocol


>This protocol is for all those that requested it and perhaps some
>on the histonet might find it interesting.
>c-Fos antibody used for immediate early response genes on thick
>free-floating sections.
>1.  intracardiac perfusion w/freshly made 4% PFA/0.1M PB
>2.  remove and store brain in 4% PFA @ 4C - overnight
>3.  store brain in 30% sucrose/0.1M PB @ 4C - overnight
>*til specimen sinks to bottom of container - if storing in this
> solution for longer time add 0.1-0.5% Thermosol to solution
> (Sigma T-5125)
>4.  cut brain on sliding microtome @ 40-50um thick and collect 
>    sections in tisue well boxes containing 0.1M PB (pH 7.4)
>Tissue sections can be stored in the following storage solution
>for very long periods @ 4C and still be used for IHC.
>    30gm sucrose
>    1gm PVP-40 (1% polyvinylpyrolidone, Sigma Cat#PVP-40)
>    30ml ethylene glycol (30%)
>    Make up the volume to 100ml w/0.1M PB pH 7.4
>5.  place sections in soaking solution using gently agitation 
>    @ RT - 2hrs
>    8.7ml of Dulbecco phosphate buffer (Sigma D-5773)
>    300ul of 10% Triton X-100
>    1ml of 1% H202 (made from 30%)
>    This makes a total of 10ml solution
>6.  wash section in DPBS X3 - 10 minutes ea.
>7.  place sections in blocking solution for  blocking nonspecific
>    binding in thick sections @ 4C w/gentle agitation - overnight
>    (17-21hrs)
>    8.5ml of IHC grade BSA (made from 2.5% BSA/300ul of 10% NA Azide/
>                            DPBS)
>*Purchased BSA from Jackson Immuno Research Labs Cat#001-000-161
>    300ul of 10% Triton X-100
>    1.5ml of Innovex background buster agent (cat#NB306 from Innovex
>                               phone #800-622-7808)
>    100ul of normal goat serum
>8.  place in c-Fos (AB-5) Oncogene cat#PC38 Lot#D10535 primary 
>    polyclonal anti rabbit antobody dilute @ 10,000/DPBS w/
>    gentle agitation @ RT - 4hrs.
>*In my experience, it's best not to switch lot numbers.
>*Oncogene phone number is 800-662-2616 or email address for customer
> inquiries: customer.service@oncresprod.com
>9.  wash in DPBS X3 - 10' ea.
>10. place in secondary antibody biotinylated goat anti-rabbit
>    (Vector BA-1000) dilute 1:1000/DPBS using gentle agitation @
>    RT - 30 mins.
>*Make ABC complex solution using Vectastain standard Elite ABC kit
> (Vector cat#PK6100)
>11. wash in DPBS X3 10' ea.
>12. incubate in ABC complex gentle agitation @ RT - 30' ea.
>13. wash well in DPBS X4 changes 10'ea.
>14. incubate section in your own DAB solution w/ or without metals
>*here can do a double DAB w/the first DAB as a pre-incubation of
> DAB and nickel (no H202) - 5-10
>*then second DAB reaction can be containing 0.01%DAB+0.5% nickel+
> 0.01% H202/DPBS - 5'
>*check sections under scope or can use dissecting scope to make sure
> your can see clearly the c-fox black or brown dots on a clean back-
> ground.
>15. wash well in DPBS X4 - 10'ea.
>16. mount sections on gelatin coated slides and air-dry - 1-2 days
>17. dehydrate in 70%,80%,95%,X4100% - 5' ea.
>    clear in X3 changes of xylene - 10'ea.
>    coverslip in permount.
>
>I hope this helps those individuals, let me know how it goes.
>regards
>Maria Mejia
>Smith-Kettlewell Eye Res. Inst
>S.F.CA
>