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Clone: BU1/75 (ICR1), Rat
Dilution: 1:400
Producer: Accurate Chemical & Scientific Corporation
Catnr.: OBT0030
Detection/Procedure
01) Deparaffinize and rehydrate sections to Automation Buffer
02) Place slides in 2N Hydrochloric Acid at 37 DegC for 30 min in water bath.
03) Place slides in Boric Acid-Borate Buffer for 1 min at room temperature.
04) Incubate slides in freshly prepared 0.01% Trypsin (in 0.5M TRIS-HCl) at 37 DegC for 3 min.
05) Rinse in distilled water for 1 min to stop digestion.
06) Rinse in 2 changes of Automation Buffer for 5 min each.
07) Quench endogenous peroxidase by placing slides in 3% hydrogen peroxide for 10 min.
08) Rinse in 2 changes of Automation Buffer for 5 min each.
09) Apply Avidin Block (A) and incubate for 15 min at room temperature.
10) Rinse slides in Automation Buffer.
11) Apply Biotin Block (B) and incubate for 15 min at room temperature.
12) Rinse slides in Automation Buffer.
13) Apply blocking serum from Vector Standard Kit. Incubate for 20 min at room temperature.
14) Tap of blocking serum.
15) Apply primary antibody and incubate for 30 min at room temperature.
16) Rinse in 2 changes of Automation Buffer for 5 min each.
17) Apply secondary antibody from Vector Standard Kit and incubate 20 min at room temperature.
18) Rinse in 2 changes of Automation Buffer for 5 min each.
19) Apply Vector ABC Reagent and incubate 30 min at room temperature.
20) Rinse in 2 changes of Automation Buffer for 5 min each.
21) Apply liquid DAB chromogen and incubate 6 min.
22) Rinse in tap water.
23) Counterstain, dehydrate and coverslip.
Reagents/Solutions
Automation Buffer:
Prepare 1L
10X Automation Buffer (Biomeda Corporation) 100 ml
Distilled Water 900 ml
Antibody Diluent:
Prepare 500 ml
Bovine serum albumin (BSA) 5 gm
1X Automation buffer 500 ml
Mix BSA with 1X automation buffer and refrigerate.
Boric Acid-Borate Buffer (pH 7.6):
Stock Solution A: Boric Acid 1.24 gm/100 ml distilled water
Stock Solution B: Sodium Biborate 1.9 gm/100 ml distilled water
Solution A ......... 85 ml
Solution B ......... 15 ml
Mix solutions A and B. Stable at room temperature.
0.5M TRIS-HCl (ph 7.8 in .1% CaCl2):
TRIS 6.057 gm
Distilled water 800 ml
CaCl2 1.0 gm
Mix TRIS, distilled water and CaCl2. pH to 7.8 with 1N HCl. Bring up to volume (1L) with distilled water.
This solution is stable at room temperature for one month.
Dako Liquid DAB Substrate-Chromogen System, DakoCytomation.
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