Difference between revisions of "Immunofluoresence"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (copied over protocol) |
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#Treat cells as required | #Treat cells as required | ||
#Wash cells twice with 2 mL cold PBS then fix for 10 min at RT | #Wash cells twice with 2 mL cold PBS then fix for 10 min at RT | ||
| − | Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanol | + | #Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanol |
#Wash twice with PBS | #Wash twice with PBS | ||
#Add 200 uL of 100mM Glycine in PBSfor 5 min to quench | #Add 200 uL of 100mM Glycine in PBSfor 5 min to quench | ||
Revision as of 00:08, 2 May 2009
Reagents
- PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)
- Neutral buffered formalin
- Cold PBS
- 100 mM Glycine in PBS
- 0.1% Triton X-100 in PBS
- Blocking Solution: 1% BSA and 1% ovalbumin in PBS
- Vectashield
Protocol
- Prepare Cells (For COS or the like, plate the day before to reach ~50%; for 3T3-L1 split one large plate into 12 wells (6 well plate, 2mL per well) at FBS day 1 and recover onto ethanol sterilized glass coverslips for 4 days in DMEM/PGS/FBS)
- Treat cells as required
- Wash cells twice with 2 mL cold PBS then fix for 10 min at RT
- Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanol
- Wash twice with PBS
- Add 200 uL of 100mM Glycine in PBSfor 5 min to quench
- Wash once with PBS
- Permeabilize for 10 min with Triton X-100 (0.1% in PBS)
- Wash three times with PBS
- Block for 1-2h with 200 uL of BSA/Ovalbumin (1% of each in PBS) blocking solution
- Incubate overnight with primary antibody in blocking solution
- Wash coverslips 3 times 10 minutes with PBS
- Incubate in 500X secondary solution
- Wash coverslips 3 times 10 minutes with PBS
- Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish
