Immunohistochemistry for AntiOsteocalcin Antibody

Immunohistochemistry for AntiOsteocalcin Antibody

(Takara, Cat #M173) Conklin Lab / Trieu Nguyen and Ed Hsiao, R. 20071102

(paraffin embedded bone sections)

REAGENTS

Primary Antibody: rabbit anti‐mouse osteocalcin (1:200) diluted in PBST/1% BSA

Secondary Antibody: donkey anti‐rabbit IgG‐HRP conjugated (1:250) diluted in PBST/1% BSA (Amersham NA9340V)

0.5% Trypsin = Lyophilized powder diluted in PBS, Sigma cat# T0303

Blocking Solution: PBSTriton (0.1%) + 2% goat serum + 1% BSA

PBS = Teknova 10X PBS diluted with water

H2O2 solution: 3% H2O2 in methanol

Hematoxylin: solution according to Mayer; Sigma 51275

DAB: DAB Substrate Kit for Peroxidase (Vector, Cat# SK‐4100)

• 5ml dH2O + 2 drops of Buffer, vortex
• Add 4 drops of DAB, vortex
• Add 2 drops Peroxide, vortex

PROTOCOL:

• Heat slides for 15 minutes at 55‐58°C (until paraffin is melted)
• Deparaffinize:

  Xylene, 5min, 2X
  100% EtOH, 5min, 2X
  90% EtOH, 5min
  70% EtOH, 5min
  dH2O, 5min

• Draw around sections with PAP pen

  Be careful not to let sections dry out

• Antigen Retrieval Process:

  Apply 0.5% trypsin to slides (0.5% = 5g/L trypsin)
  Cover with paraffin
  Incubate at 37°C for 30min in humid chamber

• Wash sections 3x 2min with dH2O
• Deactivate endogenous peroxidase with 3% H2O2 solution for 30 minutes
• Wash sections 1x 2min with PBS
• Incubate sections in blocking solution for 20min at RT
• Incubate sections in primary antibody or negative control overnight at 4°C

• Wash sections 2x 2 min with PBS
• Incubate sections in secondary antibody for 45min at RT
• Wash sections 3x 5min with PBS
• Add DAB and incubate 2‐4 minutes – stop when you notice a color change
• Rinse with dH2O for 5 min
• Apply Hematoxylin at room temperature for 0.5 min
• Dip slides in dH2O to remove excess stain
• Rinse with PBS until sections turn blue (let sit 5 min)
• Mount with Histomount