Hyperfilm, by Kodak - Product details

1

In Vitro 5-HT2A Receptor Binding

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Example 7

In Vitro Binding of 5-HT_2AReceptor

Animals:

Animals (Sprague-Dawley rats) are sacrificed and brains are rapidly dissected and frozen in isopentane maintained at −42° C. Horizontal sections are prepared on a cryostat and maintained at −20° C.

LSD Displacement Protocol:

Lysergic acid diethylamide (LSD) is a potent 5-HT_2Areceptor and dopamine D₂receptor ligand. An indication of the selectivity of compounds for either or both of these receptors involves displacement of radiolabeled-bound LSD from pre-treated brain sections. For these studies, radiolabeled ¹²⁵I-LSD (NEN Life Sciences, Boston, Mass., Catalogue number NEX-199) can be utilized; spiperone (RBI, Natick, Mass. Catalogue number s-128) a 5-HT_2Areceptor and dopamine D₂receptor antagonist, can also utilized. Buffer consists of 50 nanomolar TRIS-HCl, pH 7.4.

Brain sections are incubated in (a) Buffer plus 1 nanomolar ¹²⁵I-LSD; (b) Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar spiperone; or Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar Compound of interest for 30 minutes at room temperature. Sections are then washed 2×10 minutes at 4° C. in Buffer, followed by 20 seconds in distilled H₂O. Slides are then air-dried.

After drying, sections are apposed to x-ray film (Kodak Hyperfilm) and exposed for 4 days.

US10450276B2. Ethers, secondary amines and derivatives thereof as modulators of the 5-HT2A serotonin receptor useful for the treatment of disorders related thereto (2019-10-22). Arena Pharmaceuticals, Inc. [US]. Inventors: Bradley Teegarden [US], Dennis Chapman [US], Marc Decaire [US], Peter I. Dosa [US], Konrad Feichtinger [US], Honnappa Jayakumar [US], Thuy-Anh Tran [US], Sonja Strah-Pleynet [US], Jay Xu [US].

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2

Quantifying Receptor Binding in Mouse Brain

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Radiolabeled slides and calibrated [³H] standards were exposed to Hyperfilm (Kodak, Rochester, NY, USA) for either 10 weeks (D1 receptor) or 13 weeks (D2 receptor). Optical density measurements were made on a computerized image analyzer (MCID System, Imaging Research Inc., St. Catherines, ON, CA). Three sections were selected from the pregenual forebrain of each mouse, corresponding to Plates 17, 19 and 21 from the mouse brain atlas (Paxinos and Franklin, 2008 ). Regions of interest were traced in both hemispheres and consisted of the pregenual mPFC, dorsal striatum, and nucleus accumbens (NAcc). The sampling area of the mPFC included the cingulate, prelimbic, and infralimbic subregions. The sampling area for the NAcc included the core and shell. [³H] standards were used to generate a standard curve, and the density of radioligand bound per milligram of tissue was computed based on specific radioactivity (fmol/mg ± SEM). Specific binding was determined by subtracting non-specific binding from total binding. Data from both hemispheres were pooled since no inter-hemispheric differences were detected.

Pokinko M., Grant A., Shahabi F., Dumont Y., Manitt C, & Flores C. (2017). Dcc Haploinsufficiency Regulates Dopamine Receptor Expression Across Postnatal Lifespan. Neuroscience, 346, 182-189.

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3

In Vitro 5-HT2A Receptor Binding

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 7

In Vitro Binding of 5-HT_2AReceptor

Animals:

Animals (Sprague-Dawley rats) are sacrificed and brains are rapidly dissected and frozen in isopentane maintained at −42° C. Horizontal sections are prepared on a cryostat and maintained at −20° C.

LSD Displacement Protocol:

Lysergic acid diethylamide (LSD) is a potent 5-HT_2Areceptor and dopamine D₂receptor ligand. An indication of the selectivity of compounds for either or both of these receptors involves displacement of radiolabeled-bound LSD from pre-treated brain sections. For these studies, radiolabeled ¹²⁵I-LSD (NEN Life Sciences, Boston, Mass., Catalogue number NEX-199) can be utilized; spiperone (RBI, Natick, Mass. Catalogue number s-128) a 5-HT_2Areceptor and dopamine D₂receptor antagonist, can also utilized. Buffer consists of 50 nanomolar TRIS-HCl, pH 7.4.

Brain sections are incubated in (a) Buffer plus 1 nanomolar ¹²⁵I-LSD; (b) Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar spiperone; or Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar Compound of interest for 30 minutes at room temperature. Sections are then washed 2×10 minutes at 4° C. in Buffer, followed by 20 seconds in distilled H₂O. Slides are then air-dried.

After drying, sections are apposed to x-ray film (Kodak Hyperfilm) and exposed for 4 days.

US10781180B2. 3-phenyl-pyrazole derivatives as modulators of the 5-HT_2Aserotonin receptor useful for the treatment of disorders related thereto (2020-09-22). Arena Pharmaceuticals, Inc. [US]. Inventors: Bradley Teegarden [US], Yifeng Xiong [US], Sonja Strah-Pleynet [US], Honnappa Jayakumar [US], Peter I. Dosa [US], Konrad Feichtinger [US], Martin Casper [US], Juerg Lehmann [US], Robert M. Jones [US], David J. Unett [US], Jin Sun Karoline Choi [US].

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4

In vitro 5-HT2A Receptor Binding

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Example 14

In vitro Binding of 5-HT_2AReceptor

Animals:

Animals (Sprague-Dawley rats) are sacrificed and brains are rapidly dissected and frozen in isopentane maintained at −42° C. Horizontal sections are prepared on a cryostat and maintained at −20° C.

LSD Displacement Protocol:

Lysergic acid diethylamide (LSD) is a potent 5-HT_2Areceptor and dopamine D₂receptor ligand. An indication of the selectivity of compounds for either or both of these receptors involves displacement of radiolabeled-bound LSD from pre-treated brain sections. For these studies, radiolabeled ¹²⁵I-LSD (NEN Life Sciences, Boston, Mass., Catalogue number NEX-199) can be utilized; spiperone (RBI, Natick, Mass. Catalogue number s-128) a 5-HT_2Areceptor and dopamine D₂receptor antagonist, can also utilized. Buffer consists of 50 nanomolar TRIS-HCl, pH 7.4.

Brain sections are incubated in (a) Buffer plus 1 nanomolar ¹²⁵I-LSD; (b) Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar spiperone; or Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar Compound of interest for 30 minutes at room temperature. Sections are then washed 2×10 minutes at 4° C. in Buffer, followed by 20 seconds in distilled H₂O. Slides are then air-dried.

After drying, sections are apposed to x-ray film (Kodak Hyperfilm) and exposed for 4 days.

US09987252B2. Primary amines and derivitves thereof as modulators of the 5-HT2A serotonin receptor useful for the treatment of disorders related thereto (2018-06-05). Arena Pharmaceuticals, Inc. [US]. Inventors: Bradley Teegarden [US], Dennis Chapman [US], Juyi Choi [US], Konrad Feichtinger [US], Sangdon Han [US], Honnappa Jayakumar [US], Thuy-Anh Tran [US], Jay Xu [US], Ning Zou [US].

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5

Immunoprecipitation and Immunoblotting of eIF4E in Mouse Hippocampus

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Proteins (400–500 μg) were extracted from the hippocampus and diluted in lysis buffer for further immunoprecipitation using the CoIP kit (Pierce) protocol. Twenty microliters of A/G PLUS-Agarose beads were loaded to a column washed with coupling buffer at 90 g for 1 min. Ten micrograms of rabbit mouse ADNP antibody (Bethyl Laboratories, Montgomery, TX, USA) were added to the beads and incubated for 1 h at 24 °C. Cross-linking using 2.5 mM disuccinimidyl suberate was performed by further 1 h incubation. Then, cleared 500 μg of brain lysate were added to the column and incubated (16 h, 4 °C). To detect the eluted antigen, proteins were separated by electrophoresis on 15% acrylamide gel containing 0.1% SDS,^{10 (link)} transferred to nitrocellulose filter (Millipore, Bedford, MA, USA) and immunostained with rabbit polyclonal antibody against mouse eIF4E (1:500), (a kind gift from Professor Orna Elroy-Stein, Tel Aviv University).^{41 (link)} Proteins were visualized using enhanced chemiluminescence reagents and exposure to hyperfilm (Kodak, Petach Tiqwa, Israel). Protein bands on hyperfilm were quantified using photochromatography analysis.

Malishkevich A., Amram N., Hacohen-Kleiman G., Magen I., Giladi E, & Gozes I. (2015). Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies. Translational Psychiatry, 5(2), e501-.

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6

LSD Receptor Binding Assay

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Example 4

Animals:

Animals (Sprague-Dawley rats) are sacrificed and brains are rapidly dissected and frozen in isopentane maintained at −42° C. Horizontal sections are prepared on a cryostat and maintained at −20° C.

LSD Displacement Protocol:

Lysergic acid diethylamide (LSD) is a potent 5-HT_2Aserotonin receptor and dopamine D₂receptor ligand. An indication of the selectivity of compounds for either or both of these receptors involves displacement of radiolabeled-bound LSD from pre-treated brain sections. For these studies, radiolabeled ¹²⁵I-LSD (NEN Life Sciences, Boston, Mass.; Catalog number NEX-199) can be utilized; spiperone (RBI, Natick, Mass.; Catalog number s-128) a 5-HT_2Areceptor and dopamine D₂receptor antagonist, can also utilized. Buffer consists of 50 nanomolar TRIS-HCl, pH 7.4.

Brain sections are incubated in (a) Buffer plus 1 nanomolar ¹²⁵I-LSD; (b) Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar spiperone; or Buffer plus 1 nanomolar ¹²⁵I-LSD and 1 micromolar compound of interest for 30 min at room temperature. Sections are then washed 2×10 min at 4° C. in Buffer, followed by 20 s in distilled H₂O. Slides are then air-dried.

After drying, sections are apposed to x-ray film (Kodak Hyperfilm) and exposed for 4 days.

US10351531B2. Pyrazole derivatives as modulators of the 5-HT_2Aserotonin receptor useful for the treatment of disorders related thereto (2019-07-16). Arena Pharmaceuticals, Inc. [US]. Inventors: Yifeng Xiong [US], Martin C. Cherrier [US], Jin Sun Karoline Choi [US], Peter I. Dosa [US], Brian M. Smith [US], Sonja Strah-Pleynet [US], Brett Ullman [US], Bradley Teegarden [US].

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Hyperfilm

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6 protocols using hyperfilm

In Vitro 5-HT2A Receptor Binding

Quantifying Receptor Binding in Mouse Brain

In Vitro 5-HT2A Receptor Binding

In vitro 5-HT2A Receptor Binding

Immunoprecipitation and Immunoblotting of eIF4E in Mouse Hippocampus

LSD Receptor Binding Assay

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