Immunocytochemical Analysis of SCN Proteins

In experiment 1, triple label immunocytochemistry (ICC) was used to detect VIP, PER1, and c-FOS proteins in the SCN core. In experiment 2, double label immunocytochemistry (ICC) was used to detect VIP and PER1 protein in the core and triple label to detect PER1, c-FOS, and AVP associated neurophysin (AVP-NP) in the shell, the sub-PVN, and the PVN. Primary antibodies included: anti-VIP made in guinea pig (1:5000; Peninsula Labs, San Carlos, CA, T-5030); anti-PER1 made in rabbit (R43, 1:5000; a generous gift of David R. Weaver; LeSauter et al., 2012 (link)); anti-c-FOS made in goat (1:1000; Santa Cruz Biotech, Dallas, TX, sc-52-G); monoclonal anti-AVP associated neurophysin P45 made in mouse (1:50; ATC CRL 1798, kindly provided by Harold Gainer). Secondary antibodies, obtained from Jackson Immuno Research, West Grove, PA, were Alexa Fluor 488-conjugated AffiniPure Donkey anti Guinea Pig (DAGP; 1:300); Cy3 Donkey anti-Rabbit (DAR; 1:500); Alexa Fluor 488 Donkey anti-Goat (DAG; 1:300); and Cy5 Donkey anti-mouse (DAM; 1:300). Alexa Fluor 633 Donkey anti-Goat (DAG; 1:300) was obtained from Invitrogen.

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Manoogian E.N., Kumar A., Obed D., Bergan J, & Bittman E.L. (2018). Suprachiasmatic Function in a Circadian Period Mutant: Duper alters light-induced activation of Vasoactive Intestinal Peptide cells and PERIOD1 immunostaining. The European journal of neuroscience, 48(11), 3319-3334.

Publication 2018

Cy3 Donkey anti-Rabbit Alexa Fluor 488 Donkey anti-Goat Cy5 Donkey anti-mouse

Alexa fluor 488 Antibodies C fos C fos proteins Donkey Goat Guinea pig Immunocytochemistry Mouse Neurophysin Per1 Protein Rabbit

Corresponding Organization : University of Massachusetts Amherst

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Variable analysis

independent variables

Primary antibodies used: anti-VIP, anti-PER1, anti-c-FOS, monoclonal anti-AVP associated neurophysin P45
Secondary antibodies used: Alexa Fluor 488-conjugated AffiniPure Donkey anti Guinea Pig, Cy3 Donkey anti-Rabbit, Alexa Fluor 488 Donkey anti-Goat, Cy5 Donkey anti-mouse, Alexa Fluor 633 Donkey anti-Goat

dependent variables

Protein expression of VIP, PER1, c-FOS, and AVP associated neurophysin in the SCN core, shell, sub-PVN, and PVN

control variables

Dilution factors of primary and secondary antibodies

controls

Positive control: Not specified
Negative control: Not specified

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