Difference between revisions of "Immunofluoresence"
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==Reagents== | ==Reagents== | ||
| − | * | + | *Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton bound proteins) |
| − | + | ||
*Cold PBS | *Cold PBS | ||
*100 mM Glycine in PBS | *100 mM Glycine in PBS | ||
| − | *0.1% Triton X-100 in PBS | + | *0.1% Triton X-100 in PBS. Can use other permeabilization agents if required |
| − | *Blocking Solution: | + | *Blocking Solution: 2% BSA PBS |
*Vectashield | *Vectashield | ||
==Protocol== | ==Protocol== | ||
| − | #Prepare Cells ( | + | #Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips |
#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 ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking |
| − | + | ||
#Wash twice with PBS | #Wash twice with PBS | ||
| − | #Add 200 uL of 100mM Glycine in | + | #Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour) |
#Wash once with PBS | #Wash once with PBS | ||
| − | #Permeabilize for | + | #Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS) |
#Wash three times with PBS (5 min each) | #Wash three times with PBS (5 min each) | ||
| − | #Block for 1-2h with 200 uL of | + | #Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA) |
| − | #Incubate overnight with primary antibody in blocking solution | + | #Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x |
| − | #Wash coverslips 3 times | + | #Wash coverslips 3 times 5 minutes with PBS with gentle rocking |
| − | #Incubate in 500X secondary solution | + | #Incubate in 500X secondary solution 45 minutes in 2% BSA PBS |
| − | #Wash coverslips 3 times 10 minutes with PBS | + | #Wash coverslips 3 times 10 minutes with PBS with gentle rocking |
| − | #Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish | + | #Add 10 uL vectashield to glass slide and place cells on slide (coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C. |
*It is possible to use ImageJ to analyse [[Colocalization]] | *It is possible to use ImageJ to analyse [[Colocalization]] | ||
| + | [[Category:Immunofluoresence]] | ||
Latest revision as of 22:35, 11 July 2012
Reagents
- Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton bound proteins)
- Cold PBS
- 100 mM Glycine in PBS
- 0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
- Blocking Solution: 2% BSA PBS
- Vectashield
Protocol
- Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
- Treat cells as required
- Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
- Wash twice with PBS
- Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
- Wash once with PBS
- Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
- Wash three times with PBS (5 min each)
- Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
- Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
- Wash coverslips 3 times 5 minutes with PBS with gentle rocking
- Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
- Wash coverslips 3 times 10 minutes with PBS with gentle rocking
- Add 10 uL vectashield to glass slide and place cells on slide (coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C.
- It is possible to use ImageJ to analyse Colocalization
