BrdU-Immunoperoxidase Staining of Paraffin Tissue Sections

M. Fero Materials
Syringes, 1 mL with 36G needles
BrdU:  3-10 mg/mL in PBS
Fixative:  Methyl Carnoy's, or 4% paraformaldehyde in PBS
Paraffin processing materials
Organic solvents: Xylene, EtOH, MeOH
0.3% H2O2:  2 mL 30% H2O2 in 200 mL MeOH
Trypsin: Dilute (10x stock, -20ºC) 1/5 in PBS
2.5 N HCl: Dilute concentrated HCl 1/22 (v/v) in H2O
Mouse anti-BrDU: DAKO cat#M0744, $205/mL
Biotinylated horse anti-mouse IgG, Avidin, biotin-HRP, Horse serum: Vector, Vectastain ABC kit, #PK4002 $185
Color substrate, (make fresh): 25 mL 50 mM Tris pH8, 1 mL DAB (3,3-diaminobenzidine, 25 mg/mL stock, -20ºC), 100 µL NiCl2 (10% w/v stock, 4ºC), 10 µL 30% H2O2.
Counter stains: Methyl Green or dilute Eosin (1/4x in 80% EtOH)

BrdU Labeling
1.  Inject 0.1mL of BrdU per gm of body weight (30-100 mg/kg).  Use higher doses for short labeling times.  Label for 1-2 hours to measure the number of cells in S-phase.  Maximal staining intensity will occurr if the labeling time exceeds the duration of S-phase (6 hours)  Longer labeling (e.g. 1-7 days) does not give the number of cells in S-phase at a given time point but will give the proliferative fraction for that time period.  To label longer than 12 hours give repeated doses every 24 hours.  Use lower BrdU dose (30 mg/kg) for prolonged labelling to avoid chemotherapy-like toxcity.  Lower doses of BrdU also effects the intensity of staining and indirectly the number of cells that will be deemed "positive".

2.  Control:  The best negative control is an additional animal given no BrdU (or more formally - injected with PBS only).  This is a "no antigen" control and is superior to using "no primary antibody" as a negative control to accurately judge the level of background staining.  Use small intestine as a positive control since this gives robust labeling in the epithelial crypts even with short labeling times.

Tissue Preparation
1.  Place freshly dissected tissues in tissue cassettes and fix either in Methyl Carnoy's for 1 hour or paraformaldehyde overnight.
2.  Embed in paraffin,  and cut 4 µM sections on glass slides (see Tissue Processing protocol).

Immunostaining
In Plastic Tubs (200 mL):
3) Deparaffinize:
     Xylene 2' (x3)
     100% EtOH 2' (x3)
     70%  EtOH 2' (x1)
4) Quench endogenous peroxidase activity (optional):
     MeOH 2' (x1)
     0.3% H2O2 in MeOH 20' (x1)
5) PBS 5' (x3).

In Coplin jars (25 mL):

6) To decrease antigen masking by chromatin proteins digest in 2X Trypsin (1mg/mL in PBS) 10' (x1)

and then wash in PBS 2' (x3)

7) Denature (depurinate) the DNA with 2.5 N HCl, 37°C 15' (x1), wash in PBS 2' (x3). (0.1N NaOH can also be used but may be harsh on certain tissues).  Steps 6 and 7 may need to be altered if they destroy other antigens necessary for double immunostaining. 

In Humid Chambers (at each step use 100 µL and cover with glass slips.  To remove the slips dip the slide in a jar with PBS.  Don't pull off the slips as this will scratch the specimen):

8) Block with Horse serum (1/80 in PBS) 30' (x1). Wash in PBS 2' (x3)

9) Primary antibody 100 µL (1/200 mouse a-BrDU) (r.t. or 37ºC) 1 hr (x1). Wash (x3).

10) Secondary antibody (biotinylated horse anti-mouse IgG) 1/250, 30' (x1). Wash (x3)

11) 100 µL Avidin + Biotin-HRP mix (1/125 v/v Sol.A, 1/125 Sol B), 30' (x1). Wash (x3)

In Coplin jars (25 mL):

12) 50 mM Tris pH8 37°C (x1)

     DAB/NiCl2 Color substrate 37°C, 3' (x1).

     Rinse under H₂O tap for 5 minutes.

 

In Plastic tubs (200 mL):

13) Counter stain in methyl green for 20' or dilute Eosin (1/4x in EtOH) for 2'.

14) Dehydrate in graded ethanol:

     95% EtOH 1' (x2),

     100% EtOH 1' (x2),

     Histoclear or Xylene 1' (x2) (Methyl green is not stable in xylene)

15) Add 1-2 drops of Permount with a glass rod and cover with glass slip.  Cure overnight in fume hood.  Don't stack slides on top of each other until fully cured (2 days).