Day One: 1. Wash in PBS 2X10min after sectioning 2.

Incubate in 100ml PBS + 1ml Hydrogen Peroxide from refrigerator for 1 hour 3. Wash in PBS 3X 10min Incubate in block serum for 1 hour on the shaker as following : 3 drops of Normal Goat Serum + 10 ml PBS-t (0.4 ml Triton-X +100 ml PBS 4. Incubate in the Primary antibody for 48 hour (1:20000) in PBS-t in the refrigerator(4C) Control: PBS-t Day Three: 5. Wash in PBS-t 3X10min 6. Incubate in the Secondary antibody for 2 hours as following: 1 drop of Anti-Rabbit serum + 10ml PBS-t 7. Wash in PBS 3X10min 8. Prepare ABC solution prior 30 min of usage as following: 20ml PBS + 8 drops of A from Goat Kit + 8 drops of B from Goat Kit mix on the Vortex 9. Incubate in ABC solution for 1 hour 10. Wash in PBS 3X 10min 11. (Done by Dr. Feng) (light sensitive put foil around the tube) Incubate in DAB kit for 2min as following: 30 ml PBS +12 drops of Buffer solution +24 drops of DAB + 12 drops of H2O2 12. Wash sections in PBS 4 x 10 13. Mount the sections onto the slides- 0.1% gelatin solution (.5g Gelatin into 500ml water) 14. Dry the slides overnight 15. Counter stain slides if necessary 16. Dehydrate through 50, 70, 100, 100 EHOH each 2min, then Xylene 2X 5min 17. Coverslip

C-Fos Immunohistochemistry

Day One: 1. Wash in PBS 2X10min after sectioning 2. Incubate in 100 ml PBS + 1ml Hydrogen Peroxide from refrigerator for 1 hour 3. Wash in PBS 3X 10min 4. Incubate in block serum for 1 hour on the shaker as following : 3 drops of Normal Goat Serum (rabbit) + 10 ml PBS-t (0.4 ml Triton-X +100 ml PBS) Contain 1% BSA and 0.07% Sodium azide. 5. Incubate in the Primary antibody for 48 hour in PBS-t as following: rabbit ER alpha (1:4000) or goat ER beta (1:500) in PBS-t overnight at 4C Control: PBS-t Day three: 6. Wash in PBS-t 3X10min 7. Incubate in the Secondary antibody for 2 hours as following: 1 drop of Anti-Rabbit serum + 10ml PBS-t or Rabbit anti-goat serum 1:500 in PBS-t (add 1.5ml water to make serum) 8. Wash in PBS 3X10min 9. Prepare ABC solution prior 30 min of usage as following: 20ml PBS + 8 drops of A from Goat Kit + 8 drops of B from Goat Kit mix on the Vortex 10. Incubate in ABC solution for 1 hour 11. Wash in PBS 3X 10min 12. (light sensitive put foil around the tube)Incubate in DAB kit for 5min as following: 30 ml PBS +12 drops of Buffer solution +24 drops of DAB + 12 drops of H2O2 13. Wash sections in PBS 4 X 10 14. Mount the sections onto the slides-0.1% gelatin solution (.5g Gelatin into 500ml water) 15. Dry the slides overnight 16. Counter stain slides if necessary 17. Dehydrate through 50, 70, 100, 100 EHOH each 2min, then Xylene 2X 5min

Estrogen Receptors Immunohistochemistry

18. Coverslip