Jacobs:Tubulin Immunostaining Protocol: Difference between revisions

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When using multiple primaries at the same time, primary’s host must be of different species.
When using multiple primaries at the same time, primary’s host must be of different species.
biocompare.com is a useful resource of commercially available antibodies
biocompare.com is a useful resource of commercially available antibodies
Example: primary: rabbit anti alpha tubulin IgG, secondary donkey anti rabbit IgG Alexa 555
Example: primary: rabbit anti alpha tubulin IgG, secondary donkey anti rabbit IgG Alexa 555


Materials:
Materials:
Circular cover glass 12 mm 1.5  
*Circular cover glass 12 mm 1.5  
Triton X-100
*Triton X-100
Paraformaldehyde (PFA)
*Paraformaldehyde (PFA)
PBS
*PBS
Bovine Serum Albumin
*Bovine Serum Albumin
Prolong Anti Fade mounting medium
*Prolong Anti Fade mounting medium
Primary and secondary antibodies
*Primary and secondary antibodies
 
Preparation:
Preparation:
1) Plate cells on 12mm circular 1.5 glass coverslips the day before in 24 well plate (50’000 cells/well)
#Plate cells on 12mm circular 1.5 glass coverslips the day before in 24 well plate (50’000 cells/well)
2) Prepare 0.5% Triton X-100 in PBS (Triton X is very viscous, so prepare a few days before and make serial dilutions)
#Prepare 0.5% Triton X-100 in PBS (Triton X is very viscous, so prepare a few days before and make serial dilutions)
3) Prepare 4% Paraformaldehyde in PBS
#Prepare 4% Paraformaldehyde in PBS
a. Perform all handlings inside fume hood and wear gloves (PFA is toxic!)
#Perform all handlings inside fume hood and wear gloves (PFA is toxic!)
b. Heat PFA in PBS to 60°C on heating plate and use magnet to stirr
#Heat PFA in PBS to 60°C on heating plate and use magnet to stirr
c. PFA dissolves and solution becomes clear
#PFA dissolves and solution becomes clear
4) Prepare 3% Bovine Serum Albumin (BSA) in PBS
#Prepare 3% Bovine Serum Albumin (BSA) in PBS


Staining:
Staining:
1) Wash cells twice with prewarmed PBS
#Wash cells twice with prewarmed PBS
2) Fix cells with 500 uL 4% PFA in PBS for 15 min at RT in fume hood
#Fix cells with 500 uL 4% PFA in PBS for 15 min at RT in fume hood
3) Wash 3x with PBS (discard all washes into toxic waste container)
#Wash 3x with PBS (discard all washes into toxic waste container)
4) Permeabilize cells with 500 uL 0.5% Triton-X in PBS for 5 min
#Permeabilize cells with 500 uL 0.5% Triton-X in PBS for 5 min
5) Wash 3x with PBS
#Wash 3x with PBS
6) Block with 3% BSA in PBS for 30 min at RT
#Block with 3% BSA in PBS for 30 min at RT
7) Prepare primary antibodies (dilute in 0.5 % BSA in PBS)
#Prepare primary antibodies (dilute in 0.5 % BSA in PBS)
8) Discard blocking solution, add primary antibodies  
#Discard blocking solution, add primary antibodies  
a. Either overnight at 4°C (decreases background)
#Either overnight at 4°C (decreases background)
b. 1-2 h at room temperature
#1-2 h at room temperature
9) Wash 3x 20 min with PBS
#Wash 3x 20 min with PBS
10) Add secondary antibodies (dilute in 0.5% BSA in PBS)
#Add secondary antibodies (dilute in 0.5% BSA in PBS)
11) Wash 3x 20 min with PBS
#Wash 3x 20 min with PBS
12) Mount with ProlongAntiFade mounting medium, let it cure for 24 hours at room temperature protected from light
#Mount with ProlongAntiFade mounting medium, let it cure for 24 hours at room temperature protected from light
13) Seal the coverslips with nailpolish
#Seal the coverslips with nailpolish

Revision as of 15:21, 4 May 2010

Immunostaining Protocol FH April 2010

This is a protocol developed for staining alpha tubulin (Abcam, ab52866) and gamma tubulin (Sigma T6557) in IMCD cells, based on Attias 2010, doi:10.1159/000262317. It can be used as a reference, but depending on your antibodies you might consider other fixation protocols such as ice cold methanol or acetone. Dilutions of antibodies have to be determined by titration.

When using multiple primaries at the same time, primary’s host must be of different species.

biocompare.com is a useful resource of commercially available antibodies Example: primary: rabbit anti alpha tubulin IgG, secondary donkey anti rabbit IgG Alexa 555

Materials:

  • Circular cover glass 12 mm 1.5
  • Triton X-100
  • Paraformaldehyde (PFA)
  • PBS
  • Bovine Serum Albumin
  • Prolong Anti Fade mounting medium
  • Primary and secondary antibodies

Preparation:

  1. Plate cells on 12mm circular 1.5 glass coverslips the day before in 24 well plate (50’000 cells/well)
  2. Prepare 0.5% Triton X-100 in PBS (Triton X is very viscous, so prepare a few days before and make serial dilutions)
  3. Prepare 4% Paraformaldehyde in PBS
  4. Perform all handlings inside fume hood and wear gloves (PFA is toxic!)
  5. Heat PFA in PBS to 60°C on heating plate and use magnet to stirr
  6. PFA dissolves and solution becomes clear
  7. Prepare 3% Bovine Serum Albumin (BSA) in PBS

Staining:

  1. Wash cells twice with prewarmed PBS
  2. Fix cells with 500 uL 4% PFA in PBS for 15 min at RT in fume hood
  3. Wash 3x with PBS (discard all washes into toxic waste container)
  4. Permeabilize cells with 500 uL 0.5% Triton-X in PBS for 5 min
  5. Wash 3x with PBS
  6. Block with 3% BSA in PBS for 30 min at RT
  7. Prepare primary antibodies (dilute in 0.5 % BSA in PBS)
  8. Discard blocking solution, add primary antibodies
  9. Either overnight at 4°C (decreases background)
  10. 1-2 h at room temperature
  11. Wash 3x 20 min with PBS
  12. Add secondary antibodies (dilute in 0.5% BSA in PBS)
  13. Wash 3x 20 min with PBS
  14. Mount with ProlongAntiFade mounting medium, let it cure for 24 hours at room temperature protected from light
  15. Seal the coverslips with nailpolish
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