Immunohistochemistry for AntiOsteocalcin Antibody

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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
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