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estrogen stain
Detection of Estrogen Receptor in Formalin-Fixed, Paraffin Embedded in Mouse Tissue


Reagents:
1X Automation Buffer
3% Hydrogen Peroxide
Antibody Diluent
Citrate Buffer
DAB Chromagen
Antigen Retrieval Solution
Hematoxylin
1X Phosphate Buffered Saline

Kit and Antibody Information:

Kit used:  Vector M.O.M.˘ Kit
Vector Laboratories, Inc.
Burlingame, CA 94010
www.vectorlabs.com
1-800-227-6666
Catalog: PK2200

The Vector M.O.M.˘ kit includes reagents needed to make blocking reagent, working diluent, secondary and label antibodies.

Avidin Biotin Blocking Kit
Vector Laboratories, Inc.
Burlingame, CA 94010
www.vectorlabs.com
1-800-227-6666
Catalog #SP-2001

Primary antibody:  Mouse anti-ER (ER1D5)
Immunotech, Inc.
1-800-458-5060
Suggested dilution:  1:25
Catalog #1545

Normal Mouse Serum
Jackson Immunoresearch Laboratories, Inc.
West Grove, PA 19390
www.jacksonimmuno.com
1-800-367-5296
Catalog #015-000-001



Staining Procedure

-Positive Control Tissue:  Female reproductive tract (Vagina)

-Stain Localization:  Nuclear

Deparaffinize and hydrate slides through the following solutions.
 
 
 
Xylene 2 times 5 minutes
100% EtOH 2 times 3 minutes
95% EtOH 2 times 3 minutes
1X Automation Buffer 2 times 5 minutes

1.  Quench endogenous peroxidase by placing slides in 3% hydrogen peroxide for 15 minutes.

2. Rinse slides in 2 changes of 1X Automation Buffer for 5 minutes each.

3. Perform Heat Induced Epitope Retrieval using decloaker
        Unmasking Techniques
        Add 500 ml distilled water to the pan of the decloaker.
        Place a full rack of slides in 1X citrate buffer and place in the decloaker.
        Note:    When using a tissue tek˘ container, add 250 mls of 1X citrate buffer.
        Decloak for 5 minutes.  Pressure after 5 min.  period________________
        Depressurize for 10  minutes.
        Remove pan top and cool for 10 min.  Temp. of buffer solution______________
        Rinse in distilled water, 2x for 3 min each
 
4. Rinse slides in 2 changes of 1X Automation Buffer for 5 minutes each.

5.   Incubate sections for 1 HOUR in MOM specific IgG blocking reagent
        Made via 2.5 mls diluent plus 2 drops of Mouse IgG blocking reagent
        New kit     yes     no     Kit   name _____________    Lot #_____________
        Exp.  Date___________

6. Apply Avidin/Biotin block
        Lot#____________    New Kit   yes     no
        Apply avidin block - 15 min @ RT.
        Quick rinse in 1X AB.
        Apply biotin block - 15 min @ RT.
        No wash, wipe excess block and apply MOM diluent.

7.  Incubate tissue sections for 5 minutes in working solution of MOM diluent MOM diluent made via 600ul protein concentrate plus 7.5 mls of PBS

Wipe excess reagent from around tissue section.  DO NOT RINSE SECTIONS WITH BUFFER.
Make primary antibody dilution in Vector MOM diluent.  (600ul of protein stock in 7.5 mls PBS)

8.  Apply primary antibody (Mouse anti-estrogen receptor) at 1:25 dilution and incubate for one hour.
          Name_____________  Lot#____________________   Aliquoted     yes  /   no
          Date Aliquoted___________________

For the negative control slides, normalize the protein concentration of the normal mouse serum to the protein concentration of the primary antibody (Estrogen Receptor).  Use this to make the 1:25 dilution and incubate for one hour.

Prepare label antibody in Vector MOM diluent and incubate 30 minutes prior to use.

9.  Rinse slides in 2 changes of 1X Automation Buffer for 5 minutes each.

10.  Apply M.O.M. biotinylated anti-mouse IgG and incubate for 10 minutes
        Made via 10ul of antibody in 2.5mls of Vector MOM diluent.
        Lot#__________________ Exp Date_______________________

11.  Rinse slides in 2 changes of 1X Automation Buffer for 5 minutes each.

12. Apply Vectastain ABC Elite label for 5 minutes.  (Prepare 30 minutes before use)
        Made via 2 drops of Reagent A plus 2 drops of Reagent B in 2.5 mls diluent
        Lot#___________ Exp Date________________

13.  Rinse slides in 2 changes of 1X Automation Buffer for 5 minutes each.

14. Apply liquid Dako DAB Chromagen for 6 minutes in the dark.
          Lot #______________   Exp.  Date____________       New   kit     yes  /    no

15. Rinse in tap water 3 minutes.

16. Counterstain with Modified Harris Hematoxylin for 45 seconds.

17. Rinse in tap water until water is clear.

18. Dehydrate through the following solutions.
 
 
 
95% Ethanol 1 change 3 minutes
100% EtOH 3 changes 3 minutes
Xylene 2 changes 5 minutes

19. Coverslip
 

updated 4/7/2003

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