BrdU labelling

Sample preparation

  1. inject animals with 0.1ml BrdU labelling reagent per 10g weight at 3 weeks of age
  2. sacrifice after 2h of labelling and dissect theknee samples
  3. fix samples in 95%EtOH 5%HAc over 48h in the fridge
  4. decalcify samples in 20% EDTA pH7.4 for 2 weeks
  5. wax embed and section (6um sections)

  

Note

in order to generate statistically robust data, use 3 animals per genotype, 3 sections per animal (from different anatomically matched regions in the knee), 3 sections per slide.

  

Staining steps

 dewax in xylene 2 x 5min

.            ↓

 100% EtOH 3min

 .            ↓

90% EtOH 3min

 .            ↓

70% EtOH 3min

 .            ↓

50% EtOH 3min

 .            ↓

1x PBS (rocker) 3 x 5min (from here on, do not let the sections dry!)

 .            ↓

4M HCl (12.3ml conc HCl in 100ml) in glass pot 15min

 .            ↓

0.1M borate buffer (5x: pH8.5, 30.9g boric acid + 13.5ml 10M NaOH, to 1l with H2O) 5min

 .            ↓

1x PBS (rocker) 3 x 2min

 .            ↓

10ml 30% H2O2 in 100ml dH2O 5min

 .            ↓

1x PBS (rocker) 3 x 2min

 .            ↓

draw ImmEdge circles around sections

keep the slides in a humidified chamber during all incubations

 .            ↓

block in 40ul goat serum Dako + 960ul 1x PBS 20min

 .            ↓

1ºAb (1:100) in 1x PBS (Abcam monoclonal rat anti BrdU ab6326) 1h

 .            ↓

1x PBS (rocker) 2 x 5min

 .            ↓

prepare ABC reagent (2.5ml PBS+dropA+dropB, 30min before use)

 .            ↓

2ºAb (1:200) in 1x PBS (goat anti-rat Abcam ab6844) 20min

 .            ↓

1x PBS (rocker) 2 x 5min

 .            ↓

ABC reagent 25min

 .            ↓

1x PBS (rocker) 2 x 5min

 .            ↓

DAB (1ml buffer+dropDAB) quench in PBS 1-10min

 .            ↓

methylene green 10min

 .            ↓

3x tap water 5min

 .            ↓

95% EtOH 3min

 .            ↓

100% EtOH 5min

 .            ↓

xylene 2 x 5min

 .            ↓

mount in Vectamount, dry overnight

 

 

Data analysis

 

  1. count BrdU positive cells (brown) in the proliferative zone and present them as a percentage of all cells (green and brown) in this zone.
  2. use One Way ANOVA to statistically analyse the data.

Example: