Ab77686

The slides were deparaffinized, and heat-induced antigen retrieval was performed using Histofine (Nichirei Biosciences Inc., Tokyo, Japan). Slides were washed with Tris-buffered saline containing Tween 20 (3 × 5 min) and incubated with 10% donkey serum for 60 min. Primary antibodies or FITC-conjugated anti-mouse mast cell tryptase antibodies (1:2000, mouse monoclonal, ab2378; Abcam, Cambridge, UK) were applied to the sections and incubated overnight at 4 °C. The primary antibody used was anti-CRHR1 antibody (1:1000, goat polyclonal, ab77686; Abcam, Cambridge, UK). For double immunofluorescence labeling, we used the primary and FITC-conjugated antibodies described above, and the appropriate secondary antibody (donkey anti-goat IgG) labeled with Alexa Fluor 594 (1:1000, ab150136; Abcam). Following incubation with the antibodies, the tissues were washed with Tris-buffered saline containing Tween 20 (3 × 5 min). The slides were coverslipped with Prolong-Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Rockford, IL, USA). Fluorescent images were obtained at the Osaka City University Graduate School of Medicine Core Imaging Facility. The tissues were examined at 400× magnification using a fluorescence microscope (BX53, Olympus).

Total protein in the hypothalamus of rats and PC12 cell lysates were extracted according to the manufacturer’s protocols (Cat number: KGP3100; KeyGEN BioTECH). The proteins were separated by electrophoresis (Bio-Rad, CA, USA) and transferred onto 0.45 µM PVDF (polyvinylidene fluoride) membranes. After blocking, the membranes were incubated with antibodies at 4°C overnight and secondary antibodies for 1.5 hours at room temperature. The primary antibodies used for western blotting were as follows: CRH (1:1,000, Proteintech, 10944–1-AP), CRHR1 (1:1,000, abcam, ab77686), CRHR2 (1:1,000, abcam, ab236982), HDAC1 (1:1,000, HuaBio, ET1605-35), HDAC2 (1:1,000, HuaBio, ET1607-78), HDAC3 (1:1,000, HuaBio, ET1610-5), HDAC8 (1:1,000, HuaBio, ET1612-90), acH3 (1:1,000, Sigma, 06599), Histone 3 (H3) (1:1,000, Abways, CY6587), and GAPDH (1:5,000, Proteintech, HRP60004).