Difference between revisions of "Immunofluoresence"

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==Reagents==
 
==Reagents==
*PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)
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*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton bound proteins)
*Neutral buffered formalin
+
 
*Cold PBS
 
*Cold PBS
 
*100 mM Glycine in PBS
 
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS
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*0.1% Triton X-100 in PBS.  Can use other permeabilization agents if required
*Blocking Solution:  1%  BSA and 1% ovalbumin in PBS
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*Blocking Solution:  2%  BSA PBS
 
*Vectashield  
 
*Vectashield  
  
 
==Protocol==
 
==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)
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#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
#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 PBS for 5 min to quench (up to 1 hour)
 
#Wash once with PBS
 
#Wash once with PBS
#Permeabilize for 10 min with Triton X-100 (0.1% in PBS)
+
#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 BSA/Ovalbumin (1% of each in PBS) blocking solution
+
#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
#Incubate overnight with primary antibody in blocking solution
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#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 10 minutes with PBS (5 min each)
+
#Wash coverslips 3 times 5 minutes with PBS with gentle rocking
#Incubate in 500X secondary solution
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#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
#Wash coverslips 3 times 10 minutes with PBS (5 min each)
+
#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]]
 
[[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

  1. Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
  2. Treat cells as required
  3. Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
  4. Wash twice with PBS
  5. Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
  6. Wash once with PBS
  7. Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
  8. Wash three times with PBS (5 min each)
  9. Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
  10. Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
  11. Wash coverslips 3 times 5 minutes with PBS with gentle rocking
  12. Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
  13. Wash coverslips 3 times 10 minutes with PBS with gentle rocking
  14. 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.