Sections were blocked in StartingBlock T20 (TBS) Blocking Buffer (Thermo Scientific, Waltham, MA) for 1 hour and subsequently transferred to the primary antisera (TH: Millipore Ab318, mouse anti-TH, 1:4,000) to incubate overnight at 4 °C. Following primary incubation, tissue was incubated in the dark in secondary antisera against mouse IgG (LI-COR 926–32212, donkey anti-mouse IRDye 800CW, 1:500) for 1 hour at room temperature. Tissue was then blocked in StartingBlock T20 (TBS) Blocking Buffer (Thermo Scientific) for 1 hour at room temperature before being incubated in the primary antisera against GFP (Abcam Ab290, rabbit anti-GFP, 1:100,000) overnight at 4 °C in the dark. Following primary incubation, tissue was incubated in secondary antisera against rabbit IgG (LI-COR 925–68021, goat anti-rabbit IRDye 680LT, 1:500) for 1 hour at room temperature. Sections were mounted on subbed slides, dehydrated via ascending ethanol washes, cleared with xylene, and coverslipped with Cytoseal. Multiplexed signal intensities were imaged with both 700 and 800 nm channels in a single scan with a resolution of 42 μm and depth set to 1.0 mm using the Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE).
Ab318
Manufactured by Merck Group
Ab318 is a laboratory instrument designed for the detection and analysis of specific antibodies. The core function of this product is to provide researchers with a reliable tool for immunological studies and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared image system, by LI COR (1 mentions)
A10037, by Thermo Fisher Scientific (1 mentions)
Ds ri1, by Nikon (1 mentions)
Alexa fluor 568 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Ab290, by Abcam (1 mentions)
Vectashield hardset mounting medium, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Richard allan scientific mounting medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using ab318
1
Multiplex Immunofluorescence Imaging
2
Dual Immunofluorescence Labeling of TH and GFP
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Sections were blocked in 10% normal donkey serum for 1 hour and subsequently transferred to the primary antisera (TH: Millipore Ab318, mouse anti-TH, 1:4,000) to incubate overnight at 4 °C. Following primary incubation, tissue was incubated in the dark in secondary antisera against mouse IgG (Invitrogen A10037, Alexa Fluor 568 donkey anti-mouse, 1:500) for 1 hour at room temperature. Tissue was then blocked in 10% normal goat serum for 1 hour at room temperature before being incubated in the primary antisera against GFP (Abcam Ab290, rabbit anti-GFP, 1:100,000) overnight at 4 °C in the dark. Following primary incubation, tissue was incubated in secondary antisera against rabbit IgG (Life Technologies, Grand Island, NY; A21206, Alex Fluor 488 goat anti-rabbit IgG, 1:500) for 1 hour at room temperature. Sections were mounted on subbed slides and coverslipped with Vectashield Hardset Mounting Medium (Vector Laboratories). Images were taken on a Nikon 90i fluorescence microscope with a Nikon DS-Ri1 camera. Figures were produced in Photoshop 7.0. Brightness, saturation, and sharpness were adjusted only as necessary to best replicate the immunostaining as viewed directly under the microscope.
3
Quantification of Dopaminergic Neurons
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Paraffin tissue sections after antigen retrieval or cell cultures on coverslips were incubated with anti-TH antibody (1:500, Millipore #AB318) to determine the number of dopaminergic cells. Briefly, samples were incubated with primary antibody at 4 °C overnight in blocking TBS buffer containing 2% normal serum and 5% BSA. Samples were exposed to secondary antibody conjugated to HRP (Dako) for 1 hr at room temperature, and then HRP’s chromogenic substrate DAB (Dako) added. Color development was monitored and photographed under light microscope. Stained coronal sections and primary cells on coverslips were dehydrated, cleared by xylene, and mounted with Richard-Allan Scientific™ Mounting Medium (ThermoFisher). Four consecutive sections taken from three individual mice in each treatment group were used to quantify the average total number of dopaminergic neurons in SNpc region. Optical intensity after DAB staining of tyrosine hydroxylase in striatum was measured by ImageJ software (http://rsbweb.nih.gov/ij/plugins/track/track.html ). Dopaminergic neurons on the coverslips were counted under blinded conditions by two independent observers. Each cell culture treatment was carried out in triplicate with a minimum of three experiments.
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