Griffin:Immunohistochemistry Paraffin - Revision history

Korey Griffin at 16:57, 8 October 2012

Korey Griffin — Mon, 08 Oct 2012 16:57:59 GMT

←Older revision		Revision as of 16:57, 8 October 2012
Line 7:		Line 7:
	[[Image:IFimage2.jpg\|thumb\|right\|Mouse pancreas tissue; Red: Amylase in zymogengranules of acinar cells, and secreted amylase in ducts, Blue: DAPI (nuclear)]]		[[Image:IFimage2.jpg\|thumb\|right\|Mouse pancreas tissue; Red: Amylase in zymogengranules of acinar cells, and secreted amylase in ducts, Blue: DAPI (nuclear)]]

-	Immunohistochemistry determines protein expression profiles in tissues using ~~chromogenic~~ substrate (3,3′-Diaminobenzidine (DAB), benzene derivative). Primary antibody binds to the protein in tissue following with a conjugated peroxidase enzyme. Addition of hydrogen peroxide + DAB, catalyzes DAB to its oxidized, brown precipitate form.	+	Immunohistochemistry determines protein expression profiles in tissues using chromagenic substrate (3,3′-Diaminobenzidine (DAB), benzene derivative). Primary antibody binds to the protein in tissue following with a conjugated peroxidase enzyme. Addition of hydrogen peroxide + DAB, catalyzes DAB to its oxidized, brown precipitate form.

	The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.		The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.

Korey Griffin at 16:56, 8 October 2012

Korey Griffin — Mon, 08 Oct 2012 16:56:54 GMT

←Older revision		Revision as of 16:56, 8 October 2012
Line 6:		Line 6:

	[[Image:IFimage2.jpg\|thumb\|right\|Mouse pancreas tissue; Red: Amylase in zymogengranules of acinar cells, and secreted amylase in ducts, Blue: DAPI (nuclear)]]		[[Image:IFimage2.jpg\|thumb\|right\|Mouse pancreas tissue; Red: Amylase in zymogengranules of acinar cells, and secreted amylase in ducts, Blue: DAPI (nuclear)]]
		+
		+	Immunohistochemistry determines protein expression profiles in tissues using chromogenic substrate (3,3′-Diaminobenzidine (DAB), benzene derivative). Primary antibody binds to the protein in tissue following with a conjugated peroxidase enzyme. Addition of hydrogen peroxide + DAB, catalyzes DAB to its oxidized, brown precipitate form.

	The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.		The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.

Korey Griffin at 22:03, 25 August 2011

Korey Griffin — Thu, 25 Aug 2011 22:03:03 GMT

←Older revision		Revision as of 22:03, 25 August 2011
Line 215:		Line 215:
	*Species control (ie. incubation with normal goat IgG)		*Species control (ie. incubation with normal goat IgG)
	*Absorption control		*Absorption control
		+
		+	==DAB enhancement==
		+
		+	===Nickel===
		+
		+	DAB (0.5 mg/ml) with 0.08% nickel sulfate and 3% H2O2
		+
		+	*Volume total: 1 ml
		+	*2% nickel sulfate 40 ul
		+	*DAB Solution 1 ml
		+	*30% H2O2 10 ul
		+
		+	To make: 2% Nickel sulfate, NiSO4.6H2O: 0.9 g into 45 ml E-pure dd H2O = 2% solution. Keep in Fridge.
		+	(Dilute: 1/25 to give a 0.08% solution.)
		+
		+	'''NOTE:''' Time to stain will be more rapid. Nickel solution develops more precipitate. Blue-black ppt
		+
		+	===Copper===
		+
		+	*0.4% cupric sulfate (CuSO4 5H2O)In 0.9% NaCl
		+
		+	After DAB reaction has been completed and rinsed, immerse slides 5-20 mins, and then rinse with PBS. Darkens regular DAB color.Room temp cupric sulfate in a glass bottle is stable for many years.

	==Trouble Shooting==		==Trouble Shooting==

Korey Griffin: /* Primary Antibody */

Korey Griffin — Thu, 15 Jul 2010 21:13:26 GMT

Primary Antibody

←Older revision		Revision as of 21:13, 15 July 2010
Line 75:		Line 75:
	*PBS/FSGO 1 ml		*PBS/FSGO 1 ml
	*normal serum 100 ul		*normal serum 100 ul
-	*biotin blocking solution (Vector Labs) 4 drops	+	*OPTIONAL : biotin blocking solution (Vector Labs) 4 drops

	'''6.''' Incubate 4 oC overnight in humidified chamber.		'''6.''' Incubate 4 oC overnight in humidified chamber.

Korey Griffin: /* Primary Antibody */

Korey Griffin — Thu, 15 Jul 2010 21:10:53 GMT

Primary Antibody

←Older revision		Revision as of 21:10, 15 July 2010
Line 70:		Line 70:
	==Primary Antibody==		==Primary Antibody==

-	'''6.''' Add primary antibody @ 1:50 to vessels on slides.	+	'''5.''' Add primary antibody @ 1:50 to vessels on slides.

	For 4 slides: antibody		For 4 slides: antibody
Line 77:		Line 77:
	*biotin blocking solution (Vector Labs) 4 drops		*biotin blocking solution (Vector Labs) 4 drops

-	'''7.''' Incubate 4 oC overnight in humidified chamber.	+	'''6.''' Incubate 4 oC overnight in humidified chamber.

	==Secondary Antibody==		==Secondary Antibody==

Korey Griffin: /* Blocking */

Korey Griffin — Thu, 15 Jul 2010 21:10:36 GMT

Blocking

←Older revision		Revision as of 21:10, 15 July 2010
Line 66:		Line 66:
	'''3.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST		'''3.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST

-	'''4.''' Add blocking solution within circles; 1 hour in humidified chamber at room temperature	+	'''4.''' Add blocking solution within circles; 1 hour in humidified chamber at room temperature, then aspirate off blocking solution
-		+
-	~~'''5.''' Aspirate~~ off blocking solution	+

	==Primary Antibody==		==Primary Antibody==

Korey Griffin: /* Primary Antibody */

Korey Griffin — Thu, 15 Jul 2010 21:10:00 GMT

Primary Antibody

←Older revision		Revision as of 21:10, 15 July 2010
Line 72:		Line 72:
	==Primary Antibody==		==Primary Antibody==

-	'''8.''' Add primary antibody @ 1:50 to vessels on slides.	+	'''6.''' Add primary antibody @ 1:50 to vessels on slides.

	For 4 slides: antibody		For 4 slides: antibody
Line 79:		Line 79:
	*biotin blocking solution (Vector Labs) 4 drops		*biotin blocking solution (Vector Labs) 4 drops

-	'''9.''' Incubate 4 oC overnight in humidified chamber.	+	'''7.''' Incubate 4 oC overnight in humidified chamber.

	==Secondary Antibody==		==Secondary Antibody==

Korey Griffin: /* Blocking */

Korey Griffin — Thu, 15 Jul 2010 21:09:42 GMT

Blocking

←Older revision		Revision as of 21:09, 15 July 2010
Line 50:		Line 50:
	==Blocking==		==Blocking==

-	'''1.''' Cool slides in tub, covered, at room temp for 1 hour	+	'''1.''' Cool slides in tub, covered, at room temp for 1 hour, then transfer to tub with plain room temperature PBS, 5 min

-	~~'''2.'''~~ During 1 hour, make up 0.5% FSGO (fish skin gelatin oil) in PBS (1 500 ml bottle PBS + 2.5 ml FSGO; allow 15 min for FSGO to come out of pipet)	+	*During 1 hour, make up 0.5% FSGO (fish skin gelatin oil) in PBS (1 500 ml bottle PBS + 2.5 ml FSGO; allow 15 min for FSGO to come out of pipet)

-	'''~~3.''' Transfer to tub with plain room temperature PBS, 5 min~~	+	'''2.''' Make up avidin blocking solution:
-		+
-	~~'''4~~.''' Make up avidin blocking solution:	+

	For 1 ml:		For 1 ml:
Line 66:		Line 64:
	*Invert tube to mix		*Invert tube to mix

-	'''5.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST	+	'''3.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST

-	'''6.''' Add blocking solution within circles; 1 hour in humidified chamber at room temperature	+	'''4.''' Add blocking solution within circles; 1 hour in humidified chamber at room temperature

-	'''7.''' Aspirate off blocking solution	+	'''5.''' Aspirate off blocking solution

	==Primary Antibody==		==Primary Antibody==

Korey Griffin: /* Secondary Antibody */

Korey Griffin — Thu, 15 Jul 2010 21:07:20 GMT

Secondary Antibody

←Older revision		Revision as of 21:07, 15 July 2010
Line 88:		Line 88:

	'''1.''' Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate		'''1.''' Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate
-	'''2.''' Make up secondary antibody:	+	'''2.''' Make up secondary antibody at 1:100-1:500:

-	per ml:	+	(1:200) per ml:
	*5 ul secondary antibody		*5 ul secondary antibody
	*100 ul normal serum		*100 ul normal serum

Korey Griffin: /* Primary Antibody */

Korey Griffin — Thu, 15 Jul 2010 21:06:14 GMT

Primary Antibody

←Older revision		Revision as of 21:06, 15 July 2010
Line 74:		Line 74:
	==Primary Antibody==		==Primary Antibody==

-	'''8.''' Add primary antibody to vessels on slides.	+	'''8.''' Add primary antibody @ 1:50 to vessels on slides.

	For 4 slides: antibody		For 4 slides: antibody