Immunohistochemistry for M1Flag Antibody (Sigma, Cat

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: o *Add 0.1% Tween‐20 to primary antibody M.O.M. diluents add 2 drops of reagent B and mix. Let sit 30 minutes before use.
 * 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
 * M.O.M. Biotinylated Anti‐Mouse IgG Reagent: Dilute stock solution 1:250 in
 * VECTASTAIN Elite ABC Reagent: 2 drops of reagent A to 2.5ml TBS, mix. Then

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: o Xylene, 5min, 2X o 100% EtOH, 5min, 2X o 90% EtOH, 5min o 70% EtOH, 5min o dH2O, 5min o Be careful not to let sections dry out  Blocking Reagent at 4°C covered with parafilm
 * Heat slides for 15 minutes at 55‐58°C (until paraffin is melted)
 * Deparaffinize:
 * Draw around sections with PAP pen
 * 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

o 20 min into incubation, make ABC reagent
 * 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
 * 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