Immunohistochemistry for M1Flag Antibody (Sigma, Cat: Difference between revisions
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(New page: '''Immunohistochemistry for M1Flag Antibody (Sigma, Cat# F3040)''' (alcohol fixed paraffin embedded bone sections) Trieu Nguyen and Ed Hsiao, Conklin lab. R. 20071105 <font color="red"...) |
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* Wash sections 2x 2 min with TBS | * Wash sections 2x 2 min with TBS | ||
* Incubate sections for 5 min in working solution of '''M.O.M. Diluent''' | * 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 | * 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 | ||
30 min (humid chamber) at 37°C | |||
o 20 min into incubation, make ABC reagent | o 20 min into incubation, make ABC reagent | ||
* Wash sections 2x 5min with TBS | * Wash sections 2x 5min with TBS | ||
Latest revision as of 14:03, 2 July 2009
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
