After fixing with 4 % formalin and paraffin-embedding, villus tissues were sectioned (5 µm). The sections were incubated with primary antibodies to SP1 (1:200, Abcam, product no. ab124804, UK), CXCR5 (1:200, Abcam, ab133706, UK) or BMP8A (1:200, Abcam, product no. ab60290, UK) after blocking with 5 % (w/v) bovine serum albumin (BSA). The sections were washed 3 times with PBS and subsequently incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (CWBIO, CW2069S, China), at 37 ℃ for 1 h. The sections were stained with diaminobenzidine (DAB) (CWBIO, CW2069S, China) and mounted for bright-field microscopy (Bremen, Germany). Immunohistochemical experiments were repeated at least 3 times for each villus sample.
Cw2069s
Manufactured by CWBIO
Sourced in China
The CW2069S is a laboratory equipment product. It is designed to perform specific tasks in a laboratory setting. The core function of this product is to provide essential capabilities for scientific research and analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
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4 protocols using cw2069s
1
Immunohistochemical Analysis of Villus Tissue
2
Immunohistochemical Analysis of Tau and GSK3β
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Brain samples were fixed followed by embedding in paraffin wax. Brain sections (3um) containing the middle brain (MB), Hipp and SN were cut using a microtome (RM2235, Leica, Wetzlar, Germany). Antigen retrieval was performed by heating the sections at 120°C for 5 min and blocked using normal goat serum. Brain sections were incubated with anti- AT8 (#MN1020, Thermo Fisher Scientific, MA, USA) or phospho GSK3β (Y216) (#ab75745, Abcam plc, Cambridge, UK) overnight at 4°C followed by incubating with the second antibody. A 3, 3ʹ-diaminobenzidine (DAB) kit (CW2069S, CWBIO, Beijing, China) was used to protein visualization and couterstained with hematoxylin. Images were captured with a microscope equipped with a camera (Eclipse80, Nikon, Tokyo, Japan). The immune-reactive cells were counted using Image J version 1.46 (NIH. MA. USA).
3
Thyroid Gland Immunohistochemistry Protocol
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Animals’ blood samples were first collected by cardiac puncture after anesthesia, and then immediately sacrificed. Serum samples were isolated from the blood samples after centrifugation (1000 g , 10 min, 4℃) and stored at −80℃ for later analysis. One thyroid gland lobe was stored at −80℃ having been frozen for 10 min in liquid nitrogen. Another thyroid gland lobe was placed in 4% PFA for 12 h, and underwent gradient dehydration in 15%, 30% sucrose for 2 days. Tissue sections were cut to 2 μm thickness. Primary antibodies against BMAL1 (NB100-2288, dilution 1:500; Novus, USA) and PER2 (NB100-125, dilution 1:250; Novus) and HRP-conjugated secondary antibodies (1:2000) supplied in a DAB kit (CW2069S, CWBIO, Beijing, China) were used for immunostaining of thyroid sections. To validate the specificity of the antibodies used for immunocytochemistry, we performed primary controls (+primary antibody/−secondary antibody) and secondary controls (−primary antibody/+secondary antibody), both of which showed no positive labeling. Immunohistochemistry images were taken with Leica Aperrio Versa 8 microscope (Leica), details of quantification by IHC Profiler (20 (link)) were listed as Supplementary Data 1.
4
Immunohistochemical Analysis of Colon Tissue
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Colon tissues were collected and fixed with 4% paraformaldehyde, dehydrated and embedded into paraffin. Antigens were retrieved by heating at 95° C in 0.1M citrate buffer for 15 minutes, and then incubated in endogenous peroxidase enzymes blocking buffer for 10 minutes at room temperature. Tissue sections were blocked with PBS containing 5% BSA and 1% Triton X-100 (T8200, Solarbio Life Science, China) for 30 minutes at room temperature, and stained with IGFBP5 (1:200, 55205-1-AP, proteintech, China), FOXP3 (1:400, #12653S, Cell Signaling Technology, USA) and RORγt (1:500, bs-23110R, Bioss, China) primary antibodies at 4°C overnight. Next, tissues were incubated in goat anti-rabbit/mouse IgG and streptavidin-HRP separately for 10 minutes. Finally, staining was carried out using DAB solution (CW2069S, Cwbio, China). All images were taken under the same exposure and intensity settings.
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