IHC was performed to show NF-kB p65 nuclear translocation at 24 h after reperfusion. Briefly, the rat brains were fixed, dehydrated, and trimmed as the same method used in the immunofluorescence assay and embedded in paraffin for slicing into 5-μm-thick sections. After dewaxing and rehydrating, the sections were immersed in 0.01 M citrate buffer for antigen retrieval using the microwave for 25 min. Endogenous peroxides were blocked by 3 % H2O2 for 10 min and nonspecific antigens were then blocked with 5 % goat serum for 20 min at 37 °C. The sections were incubated with primary anti-NF-kB p65 rabbit monoclonal antibody (#8242, Cell Signaling Technology, 1:200) at 4 °C overnight, and in sham group, the primary antibody was replaced with PBS as negative control. The SABC kit (including biotinylated goat anti-rabbit secondary antibody and Strept-Avidin-Biotin Complex; SA1028, BOSTER, China) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) peroxidase substrate (AR1022, BOSTER, China) were used to detect NF-kB p65 immunostain. Images were scanned and acquired using an OLYMPUS PM20 automatic microscope (Olympus, Tokyo, Japan).
Pm20 automatic microscope
Manufactured by Olympus
Sourced in Japan
The OLYMPUS PM20 is an automatic microscope designed for laboratory use. It features automated functions for convenient operation, including automated focusing and illumination control. The PM20 is capable of producing high-quality images for various microscopy applications.
Automatically generated - may contain errors
6 protocols using pm20 automatic microscope
1
Immunohistochemical Analysis of NF-κB p65
2
Immunohistochemical Analysis of Skin Tissue
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The control and model mice were beheaded after being anesthetized with pentobarbital (1 mg/g, i.p.). After paraffin embedding, the skin tissues were then sectioned at 5 mm thickness for immunohistochemistry. Skin was fixed in 4% paraformaldehyde for 1 h blocked by 1% BSA/PBS for 2 h and then immunostained with a purified polyclonal rabbit antibody ZAP70 (1 : 100, Sigma) and then sections were washed and incubated with the secondary antibody directed against rabbit IgG (1 : 200; Dianova). Following tissue sections were incubated with Streptavidin-HRP. Several visual fields images in a tissue section were collected randomly by the OLYMPUS PM20 automatic microscope (Olympus, Tokyo, Japan).
3
Immunohistochemical Analysis of TNIK
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IHC was conducted according to the manufacturer's protocol. Briefly, the sections were deparaffinized, processed for antigen recovery, blocked in goat serum (Wuhan Boster Biological Technology, Wuhan, China) and incubated in TNIK antibody (sc-377215, mouse polyclonal antibody, 1:50) overnight at 4 °C. The next day, the samples were incubated in goat anti-mouse secondary antibody for 30 min at 37 °C. Counterstaining was carried out with Harris's hematoxylin. For negative controls, the primary antibodies were replaced with PBS. Ten visual field images were randomly obtained from every section using an OLYMPUS PM20 automatic microscope (Olympus, Japan) and TCFY-2050 (Yuancheng Inc., China) pathology system.
4
Immunohistochemical Analysis of Norbin Expression
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Immunohistochemical staining was conducted using the avidin-biotin-peroxidase complex method, according to established protocols or the manufacturer’s instructions. The primary antibody used in the experiment was mouse anti-norbin, and the secondary antibody used in the experiment was included with the indicated kit (Wuhan Boster Biological Technology, Wuhan, China). The negative controls were incubated with PBS rather than the indicated primary antibody. The images of each section were scanned and acquired by an OLYMPUS PM20 automatic microscope (Olympus, Japan) and a TCFY-2050 (Yuancheng Inc., China) pathology system. Five random visual fields in each section were imaged (5 sections in each brain). The immunohistochemical results were assessed by automatically measuring the average integrated optical density (IOD) to determine the IOD-to-field area ratio using a Motic Med 6.0 CMIAS pathology image analysis system (Beihang Motic Inc., China)19 (link). The mean of the values from 5 fields in each slide image was subsequently calculated and used to determine the differences in the immunohistochemistry results between the epilepsy and control groups.
5
Immunohistochemical Localization of pCREB
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pCREB location and expression were detected by site-specific immunohistochemical staining (Ref. 39 (link)). The primary antibody used was a rabbit polyclonal anti-pCREB antibody (1:800, Cell Signal, Boston, USA). Sections were then incubated with a biotinylated goat anti-rabbit secondary antibody (streptavidin–peroxidase kits; Zhongshan Golden Bridge, Inc, Beijing, China). Finally, sections were treated with ABC solution at 37°C for 30 min, washed with PBS, and incubated with DAB (3,3′-diaminobenzidine; Zhongshan Golden Bridge, Inc, Beijing, China) for 3 min. As a negative control, the primary and secondary antibodies were replaced with PBS. An OLYMPUS PM20 automatic microscope (Olympus, Osaka, Japan) was used to collect the images.
6
Timm Staining for Mossy Fibers
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Timm staining is based on the staining of Zn2+-containing mossy fibers by a sulfide/silver stain as described previously. Briefly, the mice were deeply anesthetized and then fixed by transcardial perfusion with 0.9% NaCl followed by 0.3% Na2S in 100 mM phosphate buffer (PB) and 4% paraformaldehyde in 100 mM phosphate buffer (PB). After perfusion, the brains were postfixed in 4% paraformaldehyde overnight at 4°C. Coronal sections were cut at 30µm on cryostat and stained for mossy fibers using Timm staining. The processing solutions were obtained from Sinopharm Chemical Reagent Co., Ltd., China and were as follows: 50% Arabic gumc, 15ml Citric acid buffer (3.825g citric acid,3.525g sodium citrate),45ml 5.67% hydroquinone, and 0.5ml 17% silver nitrate (51 (link)–53 (link)).Then stained for 40–60 min at 37°C. After rinsing, the sections were dehydrated in alcohol, cleared in xylene, and mounted on slides with permount. Timm staining was analyzed at a magnification of 40 and 100 using an OLYMPUS PM20 automatic microscope (Olympus, Japan).
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