BrdU Immunohistochemistry for Brain
IP inject 10ml/gm body weight BrdU, 1hr later sacrifice mouse, fix in Bouin's or formalin
1. Dewax and rinse in PBS x 1 for 3min
2. Block endogenous peroxidase in 10%MeOH/3%H2O2/PBS for 30min at RT and rinse in PBS x 4 x 5min
3. Prepare 0.2mg/ml pepsin/10mM HCl/PBS (PREWARM 10mM HCl/PBS)
4. Cover in pepsin solution x 20min at 37oC
5. Denature in 2M HCl x 45min at RT (37%HCl=10M)
6. Neutralize in 0.1M Na3B pH8.5 x 10min and wash in PBS x 10min
7. Encircle tissue with pap pen
8. Block with 13ml/ml normal horse serum/PBS x 30min at RT
9. Incubate with 1o antibody mouse anti-BrdU IgG1 1:50 (BD biosciences (877) 232-8995, Cat# 347580) in blocking solution at 4oC O/N, incubate control with blocking solution only
10. Allow slides to warm up x 45min at RT, remove 1o antibody, wash with PBS x 3 x 5min
11. Mix 4ml/ml normal horse serum in PBS, add 10ml/ml 2o antibody anti-mouse IgG biotinylated, incubate x 1hr at RT
12. Prepare ABC by mixing 10ml/ml A (vortex) + 10ml/ml B (vortex) in PBS, sit 30min at RT
13. Remove 2o antibody, wash with PBS x 3 x 5min
14. Incubate with ABC x 30 min at RT, remove ABC, wash with PBS x 3 x 5min
15. Prepare DAB by mixing 20ml/ml pH7.5 buffer (vortex) + 40ml/ml DAB (vortex) +
20 ml/ml house H2O
16. Incubate with DAB x 3min at RT and wash with PBS x 2 x 5min
17. Counterstain with hematoxylin and coverslip
BrdU Stock Solution
10mg/ml BrdU in PBS (do not need FdU)
store at -20oC
BrdU Working Stock Solution
dilute to 3mg/ml BrdU in PBS
store at 4oC
Pepsin Stock Solution (10mg/ml in PBS):
50mg Pepsin (Sigma, Cat# P-6887) in 5ml PBS per aliquot
store at -20oC


Cleaved caspase-3
deparaffinze/ rehydrate sections and unmask by cooking the slides 15 minutes in
Trilogy (Cell Marque, Austin TX, catalog # CMX833) in a pressure cooker. Release pressure and transfer slides into another clean hot bath of Trilogy that was in the pressure cooker at the same time.
- Wash slides with PBS 2X
- Inhibit endogenous peroxidase activity in H2O with 3%H2O2 15min
- Wash with PBS 3X
- Block 30 min in humid chamber at 37C in 5% goat serum in PBS + .05%Tween 20
- Wash briefly in PBS and add primary antibody (cleaved caspase 3(Asp 175) diluted 1:100, Cell Signaling, catalog #9661-S) in PBS +.05% Tween 20. Incubate overnight.
- Wash 5X2min with PBS +.05% Tween 20
- Incubate slides for 1 hour with anti-rabbit IgG secondary antibody diluted in PBS (10ul/ml).
- Prepare ABC reagent to sit at rt for 30 min (10ul/ml each of A and B in PBS)
- Remove secondary and wash 3X3 min
- Incubate with ABC 30 min at rt
- prepare DAB: 20ul/ml of pH7.5 buffer +40ul/ml of DAB +20ul/ml H2O2 in house water)
- Incubate 2-5 min in DAB in dark at rt
- Rinse in house water. Counterstain in histology. Coverslip.


TUNEL assay
- deparaffinize slides in xylene 2x 10min
ethanol 100% 2min
ethanol 95% 2x 2min
ethanol 70% 2min
ethanol 50% 2min
ethanol 30% 2min
H2O 3x 2min
-. incubate in 10 mM Tris pH8 / 20 mM EDTA / 10 µg protease K 10min at 37ºC
(125 µl of 20 µg/µl stock of proK for 250 ml total), rinse in H2O 5x.
- block in 10% methanol / 3% H2O2 30min at r.t.
rinse in H2O 5x, then leave in H2O 2min at room temp
- circle embryos with hydrophobic pen
- pretreat in 1x TdT buffer 2min at r.t. (optional)
- add 20-50 µl of TdT reaction mix, incubate 1 hour at 37ºC in a humid chamber
- rinse 2x then wash 3x 5min in 2xSSC – rinse 2x then wash 3x 2min in PBS
- premix A& B from the ABC kit (10 µl of each per ml of PBS, vortex between A and B)
- block in horse serum (13 µl per ml of PBS) 30min at r.t., wash 5x in PBS
- add ABC 30min at 37ºC in humid chamber, rinse in PBS 5x, wash 3x 3min
- add Tris buffer pH 7.5 (20 µl / ml in TAP water) 2min at r.t. (optional)
- add DAB/H2O2/Tris (40 µl, 20 µl, 20 µl resp / ml TAP water) in the dark at r.t., 5-30min, rinse in PBS 5x
- counterstain in methyl green 10min (filter on 3MM Whatman first), rinse in water 5min, mount in Mowiol


Ki-67 Immunohistochemistry
NOTE: Use formalin-fixed NOT Bouin's-fixed tissue
1. Dewax
2. Microwave in 1L 10mM Citrate buffer, monohydrous, pH 6.0 for 25min. Put glass container with slides in tupperware containing buffer, cover with saran wrap and poke a single hole in saranwrap
3. Run tap water in to cool x 2min
4. Wash in PBS x 5min
5. Block endogenous peroxidase in 3%H2O2/MeOH for 10min at RT and rinse in PBS x 2 x 2min
6. Encircle tissue with pap pen
7. Block in MOM Ig Blocking Reagent (36ml MOM Ig Blocking stock solution/ml PBS) x 1hr at RT and rinse in PBS x 2 x 2min
8. Incubate in MOM Diluent (80ml MOM Protein Concentrate stock solution/ml PBS) x 5min at RT
9. Incubate with 1o antibody mouse anti-Ki-67 1:200 (Novocastra, Cat# NCL-L-Ki67-MM1) in MOM Diluent solution at 4oC O/N, incubate control with MOM Diluent solution only
10. Wash with PBS x 2 x 2min
11. Incubate with 4ml/ml 2o antibody MOM biotinylated anti-mouse IgG in MOM Diluent solution x 30min at RT
12. Prepare ABC by mixing 10ml/ml A (vortex) + 10ml/ml B (vortex) in PBS, sit 30min at RT
13. Remove 2o antibody, wash with PBS x 2 x 2min
14. Incubate with ABC x 30 min at RT, remove ABC, wash with PBS x 2 x 5min
15. Prepare DAB by mixing 20ml/ml pH7.5 buffer (vortex) + 40ml/ml DAB (vortex) +
20 ml/ml house H2O
16. Incubate with DAB x 3min at RT and wash with PBS
17. Counterstain with hematoxylin (Erica's HCS program) and coverslip

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Last modified: October 10, 2005