4% paraformaldehyde fixed paraffin embedded intestine samples were section to 5 μm thickness. After deparaffinize, rehydrate, and antigen-retrieval (10 mM sodium citrate buffer, pH 6.0), sections were blocked in 5% goat serum in PBS-T (0.3% Triton X-100) for 1 hour at room temperature. Then, sections were incubated with anti-rabbit tyrosine hydroxylase antibody (1:200) with or without anti-goat UCP1 (sc-6528, 1:150, Santa Cruz) and anti-rabbit CGRP (Cat# C8298, 1:500, Sigma) prepared in fresh blocking buffer overnight at 4°C in humidified chamber. The following day, after briefly washing with PBS three times, sections were incubated with secondary antibody Alexa 488 goat anti-rabbit (1:1000) with or without Alexa 568 donkey anti-goat (1:1000) in 5% goat serum in PBS-T at room temperature for 2 hours. After 3 times PBS washing, sections were mounted with mounting medium with DAPI. Images were visualized using confocal microscopy Zeiss LSM710.
Sc 6528
Manufactured by Santa Cruz Biotechnology
Sc-6528 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for general laboratory use. The core function of Sc-6528 is to facilitate standard laboratory procedures and experiments.
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Lab products found in correlation
3 protocols using sc 6528
1
Multicolor Immunofluorescence Staining of Intestine
2
Protein Isolation and Western Blot Analysis
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For protein isolation, the medium of cells was aspirated and the plates were stored on ice. The cell monolayer was washed gently one time with ice-cold PBS. Excess PBS was aspirated. A volume of 50 µl of RIPA lysis buffer with inhibitors was added to each well of the 24-well plate. RIPA buffer was composed of 50 mM Tris-HCL pH 7.5, 150 mM NaCL 1 mM EDTA, 0.1% sodium deoxycholate, 1% NP-40, 1× protease inhibitor and 1× phosphatase inhibitor. An ice-cold cell scraper was used to scrape the cells. The lysate was transferred to 1.5 ml Eppendorf tubes. The samples were snap-frozen in liquid nitrogen and thawed on ice for three repeated cycles. After 10 min of centrifugation at 12,000× g and 4 °C the supernatant was transferred to fresh tubes and stored on −80 °C. Protein concentration was determined using the bicinchonoinic acid method. Samples were separated by SDS-PAGE after being mixed with 4× Laemmli Sample Buffer containing 10% β-mercaptoethanol and heated to 95 °C for 5 min. Afterwards, proteins were transferred to nitrocellulose membrane for incubation with primary antibodies raised against PGC-1A (sc-13067, Santa Cruz 1:500), UCP1 (sc-6528, Santa Cruz, 1:500). Calnexin (208–880, Calbiochem, 1:5000) served as loading control. Uncropped scans of the blots can be found as a Supplementary Figure in the Supplementary Information .
3
Multicolor Immunofluorescence Staining of Intestine
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