Immunofluoresence: Difference between revisions

Line 1:

==Reagents==

*Fixative: Neutral buffered formalin or 4% Paraformaldehyde in PBS or ice cold 10% methanol

*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol (for lipid/membrane 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: 1% BSA

*Blocking Solution: 2% BSA PBS

*Vectashield

==Protocol==

#Prepare Cells at required density in 12 well dishes on ethanol sterilized glass coverslips

#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

#Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)

#Wash once with PBS

#Permeabilize for 5 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)

#Block for 1-2h with 200 uL of blocking solution

#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)

#Incubate overnight with primary antibody in blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x

#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

#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. Fix with nail polish.

#Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish, dry for at least 1 hour.

*It is possible to use ImageJ to analyse [[Colocalization]]

[[Category:Immunofluoresence]]

@@ Line 1: / Line 1: @@
 ==Reagents==
-*Fixative: Neutral buffered formalin or 4% Paraformaldehyde in PBS or ice cold 10% methanol
+*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol (for lipid/membrane 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:  1%  BSA
+*Blocking Solution:  2%  BSA PBS
 *Vectashield
 ==Protocol==
-#Prepare Cells at required density in 12 well dishes on ethanol sterilized glass coverslips
+#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
+#Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
 #Wash once with PBS
-#Permeabilize for 5 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)
-#Block for 1-2h with 200 uL of blocking solution
+#Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
-#Incubate overnight with primary antibody in blocking solution at 4C.  Dilution varies with the antibody, but typically start with 200x
+#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
+#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.  Fix with nail polish.
+#Add 10 uL vectashield to glass slide and place cells on slide.  Fix with nail polish, dry for at least 1 hour.
 *It is possible to use ImageJ to analyse [[Colocalization]]
 [[Category:Immunofluoresence]]