Immunofluoresence - Revision history

Irith at 22:35, 11 July 2012

2012-07-11T22:35:59Z

← Older revision		Revision as of 22:35, 11 July 2012
Line 1:		Line 1:
	==Reagents==		==Reagents==
	*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for ~~cytoskeleton~~ bound proteins) in PBS or ice cold 10% methanol (for ~~lipid/membrane~~ bound proteins)		*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

Irith at 19:47, 23 March 2012

2012-03-23T19:47:27Z

← Older revision		Revision as of 19:47, 23 March 2012
Line 21:		Line 21:
	#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS		#Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
	#Wash coverslips 3 times 10 minutes with PBS with gentle rocking		#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, dry for at least 1 hour.		#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]]

Irith at 18:04, 22 March 2012

2012-03-22T18:04:23Z

← Older revision		Revision as of 18:04, 22 March 2012
Line 1:		Line 1:
	==Reagents==		==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		*Cold PBS
	*100 mM Glycine in PBS		*100 mM Glycine in PBS
	*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required		*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
	*Blocking Solution: 1% BSA		*Blocking Solution: 2% BSA PBS
	*Vectashield		*Vectashield

	==Protocol==		==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		#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 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 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		#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)		#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		#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		#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]]		*It is possible to use ImageJ to analyse [[Colocalization]]
	[[Category:Immunofluoresence]]		[[Category:Immunofluoresence]]

Davebrid: updated protocol

2011-08-24T15:17:14Z

updated protocol

← Older revision		Revision as of 15:17, 24 August 2011
Line 1:		Line 1:
	==Reagents==		==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 in PBS or ice cold 10% methanol
	*Neutral buffered formalin
	*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: 1% BSA ~~and 1% ovalbumin in PBS~~		*Blocking Solution: 1% BSA
	*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)~~		#Prepare Cells at required density 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
	#Wash once with PBS		#Wash once with PBS
	#Permeabilize for 10 min with Triton X-100 (0.1% in PBS)		#Permeabilize for 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
	#Incubate overnight with primary antibody in blocking solution		#Incubate overnight with primary antibody in 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		#Incubate in 500X secondary solution
	#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. Fix with nail polish.

	*It is possible to use ImageJ to analyse [[Colocalization]]		*It is possible to use ImageJ to analyse [[Colocalization]]
	[[Category:Immunofluoresence]]		[[Category:Immunofluoresence]]

Davebrid: added categories

2011-02-02T19:04:25Z

added categories

← Older revision		Revision as of 19:04, 2 February 2011
Line 26:		Line 26:

	*It is possible to use ImageJ to analyse [[Colocalization]]		*It is possible to use ImageJ to analyse [[Colocalization]]
			[[Category:Immunofluoresence]]

Davebrid: /* Protocol */ added link

2009-06-03T23:55:59Z

Protocol: added link

← Older revision		Revision as of 23:55, 3 June 2009
Line 24:		Line 24:
	#Wash coverslips 3 times 10 minutes with PBS (5 min each)		#Wash coverslips 3 times 10 minutes with PBS (5 min each)
	#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

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

Davebrid: /* Protocol */

2009-06-02T17:18:20Z

Protocol

← Older revision		Revision as of 17:18, 2 June 2009
Line 17:		Line 17:
	#Wash once with PBS		#Wash once with PBS
	#Permeabilize for 10 min with Triton X-100 (0.1% in PBS)		#Permeabilize for 10 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 BSA/Ovalbumin (1% of each in PBS) blocking solution
	#Incubate overnight with primary antibody in blocking solution		#Incubate overnight with primary antibody in blocking solution
	#Wash coverslips 3 times 10 minutes with PBS		#Wash coverslips 3 times 10 minutes with PBS (5 min each)
	#Incubate in 500X secondary solution		#Incubate in 500X secondary solution
	#Wash coverslips 3 times 10 minutes with PBS		#Wash coverslips 3 times 10 minutes with PBS (5 min each)
	#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

Davebrid at 00:08, 2 May 2009

2009-05-02T00:08:02Z

← Older revision		Revision as of 00:08, 2 May 2009
Line 12:		Line 12:
	#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 cold PBS then fix for 10 min at RT
	Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanol		#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 PBSfor 5 min to quench

Davebrid: copied over protocol

2009-05-02T00:07:44Z

copied over protocol

New page

==Reagents==
*PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)
*Neutral buffered formalin
*Cold PBS
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS
*Blocking Solution: 1% BSA and 1% ovalbumin in 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)
#Treat cells as required
#Wash cells twice with 2 mL cold PBS then fix for 10 min at RT
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
#Wash once with PBS
#Permeabilize for 10 min with Triton X-100 (0.1% in PBS)
#Wash three times with PBS
#Block for 1-2h with 200 uL of BSA/Ovalbumin (1% of each in PBS) blocking solution
#Incubate overnight with primary antibody in blocking solution
#Wash coverslips 3 times 10 minutes with PBS
#Incubate in 500X secondary solution
#Wash coverslips 3 times 10 minutes with PBS
#Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish