Livesey: Antigen retrival - Revision history

Jakob Suckale: wiki markup

2009-10-19T15:34:33Z

wiki markup

←Older revision		Revision as of 15:34, 19 October 2009
Line 1:		Line 1:
	Adapted from Hevner Protocol		Adapted from Hevner Protocol
-		+	# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
-	1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).	+	# Leave the chosen slides at room temperature just until the frost melts.
-		+	# Hydrate slides in 1x PBS for 10 seconds.
-	2. Leave the chosen slides at room temperature just until the frost melts.	+	# Put slides into '''0.01 M Citric acid (pH 6.0), microwave''' just to a boil, and let cool for 5 minutes.
-		+	# Repeat Step 4 two more times (three times total).
-	3. Hydrate slides in 1x PBS for 10 seconds.	+	# Rinse the slides 6 times in 1x TBST for 8 minutes each.
-		+	# Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
-	4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.	+	# Incubate overnight at 4ºC with primary antibodies in TBST plus 5% milk.
-		+	# Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
-	5. Repeat Step 4 two more times (three times total).	+	# Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
-		+	# Rinse 3 times in 1x TBS for 8 minutes each.
-	6. Rinse the slides 6 times in 1x TBST for 8 minutes each.	+	# Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
-		+	# Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
-	7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.	+
-		+
-	8. Incubate overnight at ~~4oC~~ with primary antibodies in TBST plus 5% milk.	+
-		+
-	9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.	+
-		+
-	~~10.~~ Add secondary antibodies in TBST for 1 hour at room temperature in the dark.	+
-		+
-	~~11.~~ Rinse 3 times in 1x TBS for 8 minutes each.	+
-		+
-	~~12.~~ Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.	+
-		+
-	~~13.~~ Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.	+
-		+
-		+
	TBST = TBS + 0.1% Triton		TBST = TBS + 0.1% Triton

DeanGriffiths at 11:32, 27 June 2006

2006-06-27T11:32:38Z

←Older revision		Revision as of 11:32, 27 June 2006
Line 1:		Line 1:
		+	Adapted from Hevner Protocol
		+
	1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).		1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).

DeanGriffiths at 11:12, 27 June 2006

2006-06-27T11:12:25Z

New page

1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).

2. Leave the chosen slides at room temperature just until the frost melts.

3. Hydrate slides in 1x PBS for 10 seconds.

4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.

5. Repeat Step 4 two more times (three times total).

6. Rinse the slides 6 times in 1x TBST for 8 minutes each.

7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.

8. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.

9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.

10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.

11. Rinse 3 times in 1x TBS for 8 minutes each.

12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.

13. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.

TBST = TBS + 0.1% Triton