Immunofluoresence: Difference between revisions

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==Reagents==

*~~PHEM Buffer~~ (~~25 mM HEPES,~~ 10 ~~mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde~~)

*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

*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: 1% BSA ~~and 1% ovalbumin in~~ PBS

*Blocking Solution: 2% BSA 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)~~

#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 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

#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

#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

#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

#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

#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]]

[[Category:Immunofluoresence]]

@@ Line 1: / Line 1: @@
 ==Reagents==
-*PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)
+*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
 *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:  1%  BSA and 1% ovalbumin in PBS
+*Blocking Solution:  2%  BSA 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)
+#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 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
-#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
-#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
+#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
+#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
+#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]]
+[[Category:Immunofluoresence]]