The hamster brain tissue was fixed in 10% buffered formalin solution, and paraffin sections (5-μm in thickness) were prepared routinely. Sections were treated with an antigen retrieval kit (Boster, AR0022) for 1 min at 37°C and quenched for endogenous peroxidases in 3% H2O2 in methanol for 10 min. After blocking in 1% normal goat serum, the sections were incubated with 1:100-diluted polyclonal antibody for REST at 4°C overnight. Rabbit isotype antibody (Zhongshan Goldenbridge, ZDR-5003) was used as the negative control. Subsequently, the sections were incubated with 1:250-diluted HRP-conjugated goat anti-rabbit secondary antibody (Zhongshan Goldenbridge, ZB-2301) for 60 min, and visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride (DAB). The slices were counterstained with hematoxylin, dehydrated and mounted on a slide and viewed under an Olympus BX51 microscope. Density analysis was performed using the Image J software (National Institutes of Health, USA). REST staining for each region was separately assessed and the mean optical density (MOD) was recorded. The OD analyses were performed by two investigators in a double-blind manner. Non-specific background correction in each section was done by subtracting the OD value of the blank area obtained from the same section.
Ar0022
Manufactured by Boster Bio
Sourced in China
The AR0022 is a high-performance CCD image sensor designed for a variety of industrial and scientific imaging applications. It features a 1.2 megapixel resolution and can capture images at up to 30 frames per second. The sensor utilizes a 5.2 μm pixel size and offers excellent low-light sensitivity and dynamic range performance.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (1 mentions)
Tris cl, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Gtx635654, by GeneTex (1 mentions)
Dab peroxidase substrate, by OriGene (1 mentions)
Af5186, by Affinity Biosciences (1 mentions)
Df6008, by Affinity Biosciences (1 mentions)
Af1009, by Affinity Biosciences (1 mentions)
Ar0005, by Boster Bio (1 mentions)
Df7561, by Affinity Biosciences (1 mentions)
Slide scanner, by 3DHISTECH (1 mentions)
Ar0009, by Boster Bio (1 mentions)
Pv 9001, by ZSGB-BIO (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
8 protocols using ar0022
1
Immunohistochemical Analysis of REST in Hamster Brain
2
Immunohistochemical Detection of Viral Antigen
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A ten percent buffered formalin solution was used to fix all the collected organs, and paraffin sections (3–4 µm in thickness) were prepared routinely as described in a previous report. Briefly, reagent from an antigen retrieval kit (Boster, AR0022) was applied to the sections at 37 °C for 1 min. A three percent solution of H2O2 in methanol was used to quench endogenous peroxidases for 10 min. The slices were incubated at 4 °C overnight with a laboratory-prepared 7D2 monoclonal antibody (1:200) after blocking in 1% normal goat serum. Horseradish peroxidase (HRP)-labelled goat anti-mouse IgG secondary antibody (Beijing ZSGB Biotechnology, ZDR-5307, 1:200) was added and maintained at 37 °C for 60 min. The slices were treated with 3,3′-diaminobenzidine tetrahydrochloride for visualization. The sections were counterstained with haematoxylin, dehydrated, mounted on a slide, and observed with an Olympus microscope. The sequential sections from all collected tissues were directly incubated with HRP-labelled goat anti-mouse IgG and used as the omission control for viral antigen staining. The sequential sections from all collected tissues were incubated with a recombinant anti-mouse IgG antibody [RM104] (Abcam, ab190475, 1:1000) as the negative control for the expression of viral antigen.
3
Immunohistochemical Analysis of Tissue Samples
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Mouse samples, rat IVD tissues and human NP samples were decalcified, dehydrated, cleared with dimethylbenzene after fixation in 4% paraformaldehyde, and specimens were embedded in paraffin. Each slice was cut into 5-μm thick sections, which were pretreated with antigen retrieval buffer (enzymatic digestion) (AR0022; Boster Biological Technology, Wuhan, China) for 30 min at 37°C. After blocking in goat serum for 30 min at room temperature, serial slices were incubated with primary antibodies (listed in Table S5
4
Immunofluorescent analysis of Nrf2 and HO-1
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The JEG-3 cells were inoculated on the petri dishes with polylysine, and then were fixed with 4% paraformaldehyde. Afterward, antigen retrieval solution (Cat. No.: AR0022, Boster, Wuhan, China) was added, followed by washing with PBS for at least three times. The goat serum blocking buffer (S-1000-20, Vector Lab, Hongkong, China) was added to the mixture, followed by incubation at 37°C for 30 min. The mixture was then incubated with Nrf2 and HO-1 monoclonal antibody (1:100). Subsequently, the goat-anti-mice secondary antibodies were added. 4',6-Diamidino-2-phenylindole was used for the nuclear staining. Finally, the images were observed under a fluorescence microscope (BX51, Olympus, Japan).
5
Histopathological Analysis of SARS-CoV-2 Infection
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A 10% buffered formalin solution was prepared to fix all the collected tissues and organs. Paraffin sections (3–4 mm in thickness) were cut following established procedures. All the lung sections were stained with hematoxylin and eosin. The histopathological observation was carefully recorded by observing slides using an Olympus microscope. For IHC staining, to identify the expression of SARS-CoV-2 S protein (GTX635654, 1:200; GeneTex, Irvine, CA), dehydrated paraffin sections (3–4 µm in thickness) were treated with an antigen retrieval kit (AR0022; Boster Bio, Pleasanton, CA) and quenched for endogenous peroxidases in three percent H2O2 in methanol for 10 min. After blocking in 1% normal goat serum for 1 h at 37 °C, the sections were stained with anti-SARS-CoV-2 rabbit monoclonal antibody at 4 °C overnight, followed by incubation with a horseradish peroxidase HRP-labeled goat anti-rabbit IgG secondary antibody (ZDR-5306, 1:200; ZSGB Bio) for 1 h at 37 °C. Finally, the sections were visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and viewed carefully by an Olympus microscope.
6
Immunohistochemical Analysis of Murine Knee Cartilage
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Knee joint sections of mice were deparaffinized and moved in 2 changes of xylene each for 10 min, hydrated in a series of ethanol (100% ethanol, 95% ethanol, followed by one shift of 70% ethanol), and rinsed twice in PBS. The antigen was retrieved by retrieval kit (AR0022; Boster) for the sections (30min; 37 °C). H2O2 (0.5%) was used to quench endogenous peroxidases for 10 min. The tissue sections were incubated with selected primary antibodies (overnight; 4 °C). Slides were rinsed in PBS containing 0.1% tween 20 (PBST, catalog P1397, Millipore Sigma) and antigen blocked with 5% normal goat serum. Next, overnight incubation at 4 °C with rabbit anti-mouse ACAN (1:200 dilution, catalog DF7561, Affinity), mouse anti-mouse Runx2 (1:200 dilution, Affinity; AF5186), rabbit anti-mouse MMP-13 (1:200 dilution, catalog 18165-1-AP, Proteintech), HIF-1α (1:200 dilution, Affinity; AF1009) and ATF4 (1:200 dilution, Affinity; DF6008). Slides were incubated with anti-rabbit secondary antibody for 30 min at room temperature, 3 times PBS rinsed for 5 min each, followed by two washes in deionized water for 5 min each. A 3-min incubation detected antibody binding to ACAN, MMP13, Runx2, HIF-1α, and ATF4 antigen with DAB peroxidase substrate (catalog zli90181, OriGene). Mayer's hematoxylin solution (catalog AR0005, BOSTER) was used to counterstain nuclei for 10–15 s.
7
Femur Immunohistochemistry of CXCR2 and CD79a
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The femurs from the two groups at aged 22 weeks were fixed in 4% paraformaldehyde for 24 h. Then, the femur sections (5 μm) were decalcified, dehydrated, and paraffin-embedded using standard histologic techniques. Endogenous peroxidases were blocked with 3% H2O2. Next, antigen retrieval was completed with a complex digestive solution (AR0022; Boster), blocked with goat serum (AR0009; Boster), and then stained with CXCR2 (1:500; PAB18401; Abnova) and CD79a (1:500; RAB00679; Abnova) at 4 °C overnight. The sections were incubated with enhanced enzyme-labeled goat anti-rabbit IgG polymer (PV-9001; ZSGB-BIO), and the color reaction was conducted using DAB (ZLI-9018; ZSGB-BIO). The sections were scanned by a slide scanner using specific software (3DHISTECH, Ltd. Budapest, Hungary). The experiment was repeated more than three times.
8
Immunohistochemical Analysis of Mouse Knee Joints
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Mouse knee joint sections evaluated by IHC were depara nized in 2 changes of xylene for 10 minutes each, rehydrated in a series of ethanol (100% ethanol, followed by 95% ethanol, followed by 1 change of 70% ethanol), and rinsed twice in deionized water. An antigen retrieval kit (AR0022; Boster) was prepared for the sections for 1 minute at 37°C.
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