Griffin:Immunofluorescence Cell Staining - Revision history

Korey Griffin at 17:30, 29 March 2013

2013-03-29T17:30:05Z

←Older revision		Revision as of 17:30, 29 March 2013
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	==CruzFluor™ Excitation/Emission==		==CruzFluor™ Excitation/Emission==
		+
		+	[[Image:CruzFluor.jpg\|thumb\|right\|Bleaching and photostable are relative; CruzFluor are comparable to Alexa.]]

	*CruzFluor™ 350: Ex (nm) 345 Em (nm) 442		*CruzFluor™ 350: Ex (nm) 345 Em (nm) 442

Korey Griffin at 00:04, 16 February 2013

2013-02-16T00:04:17Z

←Older revision		Revision as of 00:04, 16 February 2013
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	[http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf U Toronto Wright Cell Imaging Facility]		[http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf U Toronto Wright Cell Imaging Facility]

-	~~[[Category~~:~~Protocol]] [[Category~~:~~In vitro]] [[Category~~:~~Mammalian cell culture]]~~	+	==CruzFluor™ Excitation/Emission==
		+
		+	*CruzFluor™ 350: Ex (nm) 345 Em (nm) 442
		+	*CruzFluor™ 405: Ex (nm) 401 Em (nm) 420
		+	*CruzFluor™ 488: Ex (nm) 491 Em (nm) 514
		+	*CruzFluor™ 514: Ex (nm) 518 Em (nm) 542
		+	*CruzFluor™ 532: Ex (nm) 542 Em (nm) 558
		+	*CruzFluor™ 555: Ex (nm) 559 Em (nm) 569
		+	*CruzFluor™ 594: Ex (nm) 592 Em (nm) 614
		+	*CruzFluor™ 633: Ex (nm) 638 Em (nm) 655
		+	*CruzFluor™ 647: Ex (nm) 654 Em (nm) 674
		+	*CruzFluor™ 680: Ex (nm) 682 Em (nm) 701
		+	*CruzFluor™ 700: Ex (nm) 693 Em (nm) 713
		+	*CruzFluor™ 750: Ex (nm) 753 Em (nm) 779
		+	*CruzFluor™ 790: Ex (nm) 782 Em (nm) 811

Korey Griffin: /* Cell Culture fixatives for optimization */

2011-10-20T00:13:11Z

Cell Culture fixatives for optimization

←Older revision		Revision as of 00:13, 20 October 2011
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	==Cell Culture fixatives for optimization==		==Cell Culture fixatives for optimization==

-	[[Image:~~IFimage2~~.jpg\|thumb\|right\|Granular nuclear localization with phase contrast overlay]]	+	[[Image:IFimage3.jpg\|thumb\|right\|Granular nuclear localization with phase contrast overlay]]

	===Methanol fixation (for cytoskeletal components)===		===Methanol fixation (for cytoskeletal components)===

Korey Griffin at 00:12, 20 October 2011

2011-10-20T00:12:41Z

←Older revision		Revision as of 00:12, 20 October 2011
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	==Cell Culture fixatives for optimization==		==Cell Culture fixatives for optimization==
		+
		+	[[Image:IFimage2.jpg\|thumb\|right\|Granular nuclear localization with phase contrast overlay]]

	===Methanol fixation (for cytoskeletal components)===		===Methanol fixation (for cytoskeletal components)===

Korey Griffin: /* Cell Adhesion Enhancement */

2010-04-30T16:43:16Z

Cell Adhesion Enhancement

←Older revision		Revision as of 16:43, 30 April 2010
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	==Cell Adhesion Enhancement==		==Cell Adhesion Enhancement==
		+
		+	Low adhesive phenotype or suspension type cells may require additional care for labeling on a solid phase.

	===Poly-D-lysine (PDL)/Collagen===		===Poly-D-lysine (PDL)/Collagen===

Korey Griffin at 16:42, 30 April 2010

2010-04-30T16:42:15Z

←Older revision		Revision as of 16:42, 30 April 2010
Line 51:		Line 51:
	*Rinsed with PBS.		*Rinsed with PBS.

-	==Frozen tissue fixatives for optimization==	+	==Frozen tissue section fixatives for optimization==

	===Acetone fixation===		===Acetone fixation===
Line 61:		Line 61:
	*Fix slides in cold acetone (-20oC) for 10 minutes		*Fix slides in cold acetone (-20oC) for 10 minutes
	*Rinse three times with PBS		*Rinse three times with PBS
		+
		+	==Cell Adhesion Enhancement==
		+
		+	===Poly-D-lysine (PDL)/Collagen===
		+
		+	Poly-D-lysine (PDL) or collagen coated plates/slides can enhance cell binding due to the creation of a uniform net positive charge. This type of coating can enhance cell attachment, growth and differentiation. PDL coated surfaces can reduce cell detachment from shear stress in washing steps associated with cell labeling.
		+
		+	*Promotes cell plating efficiency and improves morphology
		+	*Uniform net positive charge on plastic surface enhances cell attachment
		+
		+	===Cytospin===
		+
		+	Suspension cells can be centrifuged onto glass slides. [http://www.ihcworld.com/_protocols/histology/cytospin.htm Cytospin] technique utilizes a single layer of cells that are deposited onto a clearly-defined area of a glass slide. The slides are designed such that a "filter card" absorbs the excess fluid from the cells. This technique deposits the cells for nuclear presentation.
		+
		+	Cytospin has many variables; rpm, time of spinning, and cell concentration - and these variables will determine the quality and cell morphology of the prep. For this reason, Smearing suspension cells onto coated slides may be a favorable technique.

	==DAPI Counterstain==		==DAPI Counterstain==

Korey Griffin: /* Formaldehyde fixation (for membrane associated components) */

2010-03-03T20:20:29Z

Formaldehyde fixation (for membrane associated components)

←Older revision		Revision as of 20:20, 3 March 2010
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	===Formaldehyde fixation (for membrane associated components)===		===Formaldehyde fixation (for membrane associated components)===
-	Higher polymers, which are initially insoluble, are sold as a white powder known as paraformaldehyde.	+	Higher polymers, which are initially insoluble, are sold as a white powder known as paraformaldehyde. The concentration of formaldehyde in compound fixatives varies widely - ranging from 0.5 to 15% w/v. The majority of fixatives using formaldehyde have a concentration of between 2.5 and 4% w/v.

	*Rinse cells with PBS at room temperature.		*Rinse cells with PBS at room temperature.

Korey Griffin: /* Tissue/Cell Autofluorescence */

2010-02-12T23:46:29Z

Tissue/Cell Autofluorescence

←Older revision		Revision as of 23:46, 12 February 2010
Line 168:		Line 168:
	*Rinse many times with PBS to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it loses its reactivity with time.		*Rinse many times with PBS to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it loses its reactivity with time.

-	[http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf U Toronto Wright Imaging Facility]	+	[http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf U Toronto Wright Cell Imaging Facility]

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]

Korey Griffin: /* Tissue/Cell Autofluorescence */

2010-02-12T23:46:11Z

Tissue/Cell Autofluorescence

←Older revision		Revision as of 23:46, 12 February 2010
Line 168:		Line 168:
	*Rinse many times with PBS to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it loses its reactivity with time.		*Rinse many times with PBS to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it loses its reactivity with time.

-		+	[http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf U Toronto Wright Imaging Facility]

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]

Korey Griffin at 23:44, 12 February 2010

2010-02-12T23:44:47Z

←Older revision		Revision as of 23:44, 12 February 2010
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	#Paper1 pmid=11561004		#Paper1 pmid=11561004
	</biblio>		</biblio>
		+
		+	==Tissue/Cell Autofluorescence==
		+
		+	Autofluorescence can be due to “natural fluorescence” or “fixative-induced fluorescence”. The emission spectra of natural fluorescence and fixation-induced fluorescence is broad compared to the spectra of the flurochrome dyes and probes, making it
		+	a challenge to effectively differentiate specific 'signal' from 'noise' fluorescence by traditional filtering methods. Some of the options to consider are;
		+
		+	*Avoid it by screening different flurochrome filters for the most ideal channel
		+	*Try to filter it out during image acquisition; difficult due to the broad emission spectrum.
		+	*Chemically remove it; this may also limit true signal
		+
		+	===Natural fluorescence===
		+
		+	Natural fluorescence can arise from substances like flavins and porphyrins. These compounds can be extracted by organic solvents and aren't much of a problem in fixed, dehydrated sections. There appears to be some "background" fluorescence in tissues if the laser aperture is high enough, particularly in broad-band blue excitation. Narrow-band filters and lower wattage (50 rather than 200) mercury lamps can reduce this.
		+
		+	Natural fluorescence can be helpful in highlighting tissue structure.
		+
		+	====Lipofuscin====
		+
		+	Lipofuscin is the breakdown product of old red blood cells – an “aging” pigment. It usually occurs as small, punctate intracellular structures that are strongly fluorescent under any excitation ranging from 360nm to 647 nm. The colour should appear orange under UV excitation, green or yellow under blue excitation, or red under green excitation. Lipofuscin, should be less prevalent in younger (e.g., early adult) animals.
		+
		+	====Elastin and Collagen====
		+
		+	Typically from blood vessel walls. Elastin contains several fluorophores. There is a similar fluorophore to that found in collagen.Nearly all vessels on the arterial side of the circulation have an internal elastic lamina that lies between the endothelium and the innermost layer of smooth muscle cells. Elastin is fluorescent over a range of excitation wavelengths.
		+	Most of the extracellular material between the smooth muscle and in the adventitia layer is collagen which is also autofluorescent.
		+
		+	===Fixative-induced Fluorescence===
		+
		+	Aldehyde fixatives react with amines and proteins to generate fluorescent products in long-pass green emission (GFP, FITC, Alexa488). Glutaraldehyde is more intense than formaldehyde. The simplest way to stop aldehyde-induced fluorescence is to use Formalin or PFA rather then glutaraldehyde.
		+
		+	Glutaraldehyde exists as low polymers that reacts with and cross-links protein molecules,and produces free aldehyde groups in the process. These tissue-bound free aldehyde groups will combine covalently with any amino group offered to them, including terminal and side-chain (lysine) amino groups of proteins being used as histochemical reagents; primary antibodies, all lectins and all enzymes. A highly specific monoclonal primary antibody may bind at sites that contain basic proteins but not the antigen you're after.
		+
		+	Aldehyde-induced fluorescence in summary is due to reaction of free aldehydes with tissue components. Glutaraldehyde is more prominent than formaldehyde; and the effect is compounded when fixation is longer or warmer. Fairly uniform, non-punctate fluorescence distribution across tissue may be brighter in some cells than others depending upon the presence of biogenic amines.
		+
		+	====Aldehyde Blocking====
		+
		+	Aldehyde Blocking can minimize aldehyde-induced fluorescence by reducing the -CHO groups to -OH with sodium borohydride.
		+
		+	*Immediately before use, make up a 1 mg/ml solution of sodium borohydride in a physiological buffer such as PBS. The solution will be fizzy like carbonated water. Preparing this solution on ice and performing all subsequent incubations on ice has also been recommended.
		+	*Apply this solution immediately (while fizzing) to cells or tissue sections. For glutaraldehyde fixed cell monolayers incubate in the sodium borohydride solution for 4 minutes. Replace with fresh sodium borohydride solution for another 4 minutes. For paraformaldehyde fixed paraffin embedded 7 µm sections incubate 3 times, 10 minutes each in sodium borohydride solution. For thicker tissue sections, more changes of sodium borohydride solution and/ or longer periods of incubation might be necessary.
		+	*Rinse many times with PBS to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it loses its reactivity with time.
		+
		+

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]