Immunohistochemistry for M1Flag Antibody (Sigma, Cat

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Immunohistochemistry for M1Flag Antibody (Sigma, Cat# F3040)

(alcohol fixed paraffin embedded bone sections)

Trieu Nguyen and Ed Hsiao, Conklin lab. R. 20071105


REAGENTS VECTOR M.O.M. Immunodetection Kit (Peroxidase) (Vector, Cat# PK‐2200)

WORKING SOLUTIONS:

  • M.O.M. Mouse Ig Blocking Reagent: 2 drops of stock solution to 2.5ml TBS
  • M.O.M. Diluent*: Dilute Protein Concentrate stock solution 1:13.5 in TBS
      o *Add 0.1% Tween‐20 to primary antibody
  • M.O.M. Biotinylated Anti‐Mouse IgG Reagent: Dilute stock solution 1:250 in
        M.O.M. diluents
  • VECTASTAIN Elite ABC Reagent: 2 drops of reagent A to 2.5ml TBS, mix. Then

add 2 drops of reagent B and mix. Let sit 30 minutes before use.

TBS

  1mM CaCl2
  50mM Tris pH7.4
  0.15M NaCl2

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:
  o Xylene, 5min, 2X
  o 100% EtOH, 5min, 2X
  o 90% EtOH, 5min
  o 70% EtOH, 5min
  o dH2O, 5min
  • Draw around sections with PAP pen
  o Be careful not to let sections dry out
  • Deactivate endogenous peroxidase with 3% H2O2 solution for 30 minutes
  • Wash sections 2x 2min with TBS
  • Incubate sections overnight (humid chamber) in working solution of M.O.M. Mouse Ig
  Blocking Reagent at 4°C covered with parafilm


  • Place slides at RT for 30min.
  • Wash sections 2x 2 min with TBS
  • Incubate sections for 5 min in working solution of M.O.M. Diluent
  • Tip excess diluent off sections. Dilute primary antibody in M.O.M. Diluent (+0.1% Tween‐20) to the appropriate concentration (1:250 for anti‐Flag). Incubate sections for 30 min (humid chamber) at 37°C
  o 20 min into incubation, make ABC reagent
  • Wash sections 2x 5min with TBS
  • Apply working solution of M.O.M. Biotinylated Anti‐Mouse IgG Reagent and incubate for 30 min
  • Wash sections 2x 2min with TBS
  • Incubate sections in ABC Reagent for 10 min
  • Wash sections 2x 5min with TBS
  • Add DAB and incubate 2‐4 minutes – stop when you notice a color change
  • Rinse with dH2O for 5 min. Checking for color change. If not enough color is apparent, reapply DAB and rinse in dH2O before the next step
  • Apply Hematoxylin at room temperature for 15‐30 seconds
  • Dip slides in dH2O to remove excess stain
  • Rinse with TBS (pH 7.4) until sections turn blue (let sit 5 min)
  • Mount with Histomount
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