AMPAR Tracking - Revision history

Monakhos at 12:49, 12 May 2013

2013-05-12T12:49:33Z

Monakhos: /* Single-Nanoparticle Tracking. */

2013-05-02T08:05:21Z

Single-Nanoparticle Tracking.

← Older revision		Revision as of 01:05, 2 May 2013
Line 272:		Line 272:

	===Single-Nanoparticle Tracking. ===		===Single-Nanoparticle Tracking. ===
	A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated (see also [http://en.wikipedia.org/wiki/Polyclonal_antibodies antibodies] to goat (Fab‘)2 anti-mouse IgG ([http://products.invitrogen.com:80/ivgn/en/US/direct/invitrogen?cmd=catProductDetail&productID=Q11422MP Invitrogen]) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.		A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated (see also [http://en.wikipedia.org/wiki/Polyclonal_antibodies antibodies]) to goat (Fab‘)2 anti-mouse IgG ([http://products.invitrogen.com:80/ivgn/en/US/direct/invitrogen?cmd=catProductDetail&productID=Q11422MP Invitrogen]) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.

	===Antibodies and drugs.===		===Antibodies and drugs.===

Monakhos: /* Single-Nanoparticle Tracking. */

2013-05-02T08:04:56Z

Single-Nanoparticle Tracking.

← Older revision		Revision as of 01:04, 2 May 2013
Line 272:		Line 272:

	===Single-Nanoparticle Tracking. ===		===Single-Nanoparticle Tracking. ===
	A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated to goat (Fab‘)2 anti-mouse IgG ([http://products.invitrogen.com:80/ivgn/en/US/direct/invitrogen?cmd=catProductDetail&productID=Q11422MP Invitrogen]) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.		A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated (see also [http://en.wikipedia.org/wiki/Polyclonal_antibodies antibodies] to goat (Fab‘)2 anti-mouse IgG ([http://products.invitrogen.com:80/ivgn/en/US/direct/invitrogen?cmd=catProductDetail&productID=Q11422MP Invitrogen]) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.

	===Antibodies and drugs.===		===Antibodies and drugs.===

Monakhos: /* MAKING QUANTUM DOTS */

2013-05-02T07:09:55Z

MAKING QUANTUM DOTS

← Older revision		Revision as of 00:09, 2 May 2013
Line 268:		Line 268:
	*[http://www.bdbiosciences.com/ecat/txDetailedTable.jsp?from=simpleTable&action=SELECT&form=formTree_catBean&item=745783&size=20&wcm_page=/research/neuroscience/productlist/reagentsassays.jsp&wcm_title=Research%20Applications%20-%20Neuroscience%20-%20Product%20List%20-%20Reagents%20and%20Assays&wcm_category=745783 BD Biosciences]		*[http://www.bdbiosciences.com/ecat/txDetailedTable.jsp?from=simpleTable&action=SELECT&form=formTree_catBean&item=745783&size=20&wcm_page=/research/neuroscience/productlist/reagentsassays.jsp&wcm_title=Research%20Applications%20-%20Neuroscience%20-%20Product%20List%20-%20Reagents%20and%20Assays&wcm_category=745783 BD Biosciences]
	*[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods Choquet Methods]		*[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods Choquet Methods]
			[http://www.bdbiosciences.com/ptProduct.jsp?prodId=29038&catyId=745783&page=product&wcm_page=/research/neuroscience/productlist/reagentsassays.jsp&wcm_title=Research%20Applications%20-%20Neuroscience%20-%20Product%20List%20-%20Reagents%20and%20Assays&wcm_category=745783 GluR2 Primary Antibody]

Monakhos: /* MAKING QUANTUM DOTS */

2013-05-02T06:54:53Z

MAKING QUANTUM DOTS

← Older revision		Revision as of 23:54, 1 May 2013
Line 266:		Line 266:

	*[https://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot/qdot_reg__nanocrystal.html Invitrogen Quantum Dots]		*[https://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot/qdot_reg__nanocrystal.html Invitrogen Quantum Dots]
			*[http://www.bdbiosciences.com/ecat/txDetailedTable.jsp?from=simpleTable&action=SELECT&form=formTree_catBean&item=745783&size=20&wcm_page=/research/neuroscience/productlist/reagentsassays.jsp&wcm_title=Research%20Applications%20-%20Neuroscience%20-%20Product%20List%20-%20Reagents%20and%20Assays&wcm_category=745783 BD Biosciences]
	*[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods Choquet Methods]		*[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods Choquet Methods]

Monakhos: /* Antibodies and drugs. */

2013-05-02T06:52:39Z

Antibodies and drugs.

← Older revision		Revision as of 23:52, 1 May 2013
Line 274:		Line 274:
	===Antibodies and drugs.===		===Antibodies and drugs.===
	[[File:Fab.png\|thumb\|200px]]		[[File:Fab.png\|thumb\|200px]]
	The antibody to the [http://www.ncbi.nlm.nih.gov/pubmed/7993626 N-terminal epitope of the GluR1 subunit] was described previously46. We used a commercial antibody to an N-terminal epitope of the GluR2 subunit to detect GluR2 (BD Pharmigen).		The antibody to the [http://www.ncbi.nlm.nih.gov/pubmed/7993626 N-terminal epitope of the GluR1 subunit] was described previously46. We used a commercial antibody to an N-terminal epitope of the GluR2 subunit to detect GluR2 ([http://www.bdbiosciences.com/ecat/ BD Pharmigen]).
	AMPA receptor labeling and synaptic live staining.		AMPA receptor labeling and synaptic live staining.
	Quantum dot 655 goat F(ab')2 antibody to rabbit IgG conjugate (H+L) highly cross-absorbed and quantum dot 655 goat F(ab')2 antibody to mouse IgG conjugate (H+L) highly cross-absorbed were obtained from Quantum Dot (Invitrogen). Receptors were stained using quantum dots pre-coated with antibody to GluR1 or monoclonal antibody to GFP. Quantum dots (0.1 M) were incubated with 1 g of antibody in 10 l of phosphate-buffered saline (PBS) for 15–30 min. Unspecific binding was blocked by adding casein (Vector Laboratories) to the pre-coated quantum dot 15 min before use. Neurons were incubated 5–10 min at 37 °C in culture medium with pre-coated quantum dots (final dilution of 0.1–0.01 nM). The incubation was followed by four washing steps of 30 s each. All incubations and washes were performed in pre-warmed extracellular HEPES-buffered solution (see below).		Quantum dot 655 goat F(ab')2 antibody to rabbit IgG conjugate (H+L) highly cross-absorbed and quantum dot 655 goat F(ab')2 antibody to mouse IgG conjugate (H+L) highly cross-absorbed were obtained from Quantum Dot (Invitrogen). Receptors were stained using quantum dots pre-coated with antibody to GluR1 or monoclonal antibody to GFP. Quantum dots (0.1 M) were incubated with 1 g of antibody in 10 l of phosphate-buffered saline (PBS) for 15–30 min. Unspecific binding was blocked by adding casein (Vector Laboratories) to the pre-coated quantum dot 15 min before use. Neurons were incubated 5–10 min at 37 °C in culture medium with pre-coated quantum dots (final dilution of 0.1–0.01 nM). The incubation was followed by four washing steps of 30 s each. All incubations and washes were performed in pre-warmed extracellular HEPES-buffered solution (see below).

Monakhos: /* MAKING QUANTUM DOTS */

2013-05-02T06:51:54Z

MAKING QUANTUM DOTS

← Older revision		Revision as of 23:51, 1 May 2013
Line 263:		Line 263:
	==MAKING QUANTUM DOTS==		==MAKING QUANTUM DOTS==

	~~[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods~~ To create a quantum dot] that will tag AMPA receptors choose a quantum dot conjugated to an [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG] antibody that also has a fragment antigen-binding region (aka F(ab’)2 or [http://en.wikipedia.org/wiki/Fragment_antigen_binding Fab] fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.		To create a quantum dot that will tag AMPA receptors choose a quantum dot conjugated to an [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG] antibody that also has a fragment antigen-binding region (aka F(ab’)2 or [http://en.wikipedia.org/wiki/Fragment_antigen_binding Fab] fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.

			*[https://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot/qdot_reg__nanocrystal.html Invitrogen Quantum Dots]
			*[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods Choquet Methods]

Monakhos: /* Single-Nanoparticle Tracking. */

2013-05-02T06:50:26Z

Single-Nanoparticle Tracking.

← Older revision		Revision as of 23:50, 1 May 2013
Line 267:		Line 267:

	===Single-Nanoparticle Tracking. ===		===Single-Nanoparticle Tracking. ===
	A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated to goat (Fab‘)2 anti-mouse IgG (Invitrogen) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.		A total of 1 μL of 655-nm Quantum dots (Qdots) conjugated to goat (Fab‘)2 anti-mouse IgG ([http://products.invitrogen.com:80/ivgn/en/US/direct/invitrogen?cmd=catProductDetail&productID=Q11422MP Invitrogen]) were incubated with 1 μL Fab anti-GluA2 in 7 μL PBS for 20 min at room temperature. Nonspecific binding was blocked by adding 1 μL of 10% casein stock solution for 15 min (Vector Laboratories), and this solution was kept at 4 °C throughout the experiment. Neurons were incubated for 10 min at 37 °C in 1 mL culture medium containing 1 μL of the anti-GluA2-coated Qdot solution, then rinsed and mounted in an aluminum chamber containing Tyrode solution (30 mM glucose, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM Hepes) on a Nikon microscope (NIKON Eclipse TE2000-U) thermostated to 37 °C using an air blower (World Precision Instruments) and an objective heater (Bioptechs). Single Qdots were detected through a100 × 1.4 N.A. oil immersion objective, using a 100-W mercury lamp and appropriate excitation/emission filters (Chroma). Sequences of 50 s, corresponding to stacks of 1,000 images with an integration time of 50 ms, were acquired using a CCD camera (Quantem; Roper Scientific). For each coverslip, Qdots were followed on randomly selected dendritic regions (size 20 × 20 μ m) for up to 20 min. Two coverslips of each condition were processed for a total of 3 – 4 neuronal cultures. We checked for specificity of Qdot binding by preparing primary hippocampal cultures from GluA2 KO mice dissected at p0. Anti – GluA2- conjugated Qdots bound minimally to cultures from GluA2 KO compared with those prepared from wild-type littermates.


	===Antibodies and drugs.===		===Antibodies and drugs.===

Monakhos: /* MAKING QUANTUM DOTS */

2013-05-02T06:48:41Z

MAKING QUANTUM DOTS

← Older revision		Revision as of 23:48, 1 May 2013
Line 263:		Line 263:
	==MAKING QUANTUM DOTS==		==MAKING QUANTUM DOTS==

	[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods To create a quantum dot] that will tag AMPA receptors choose a quantum dot conjugated to an [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG] antibody that also has a fragment antigen-binding region (aka F(ab’)2 or Fab fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.		[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods To create a quantum dot] that will tag AMPA receptors choose a quantum dot conjugated to an [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG] antibody that also has a fragment antigen-binding region (aka F(ab’)2 or [http://en.wikipedia.org/wiki/Fragment_antigen_binding Fab] fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.

Monakhos: /* MAKING QUANTUM DOTS */

2013-05-02T06:47:14Z

MAKING QUANTUM DOTS

← Older revision		Revision as of 23:47, 1 May 2013
Line 263:		Line 263:
	==MAKING QUANTUM DOTS==		==MAKING QUANTUM DOTS==

	[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods To create a quantum dot] that will tag AMPA receptors choose a quantum dot conjugated to an IgG antibody that also has a fragment antigen-binding region (aka F(ab’)2 or Fab fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.		[http://www.nature.com/neuro/journal/v12/n7/full/nn.2338.html#online-methods To create a quantum dot] that will tag AMPA receptors choose a quantum dot conjugated to an [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG] antibody that also has a fragment antigen-binding region (aka F(ab’)2 or Fab fragment, the part of an antibody that binds antigens). This entire construct, QD-IgG-Fab, is your secondary antibody construct and the IgG and Fab are usually from different host animals. This is then incubated with primary antibody fragment (Fab) that is specific to the protein of interest, in this case AMPA receptors.