After deparaffinization and rehydration, sections were soaked in sodium citrate buffer for heat-induced epitope exposure. Nonspecific binding sites were blocked by incubating with 10% goat serum at 37 °C for 1 h. Then, sections were incubated with anti–Ki-67 antibody (1:200; ab15580, Abcam, Cambridge, CA, USA) overnight at 4 °C, followed by incubation with biotinylated goat anti-rabbit IgG secondary antibodies (1:300, A0277, beyotime Co., Ltd., Shanghai, China) for 2 h. Following the washing, the sections were incubated sequentially with 1:300 HRP-streptavidin (1:300, A0303, beyotime Co., Ltd., Shanghai, China) for 2 h. After, they were treated with diaminobenzidine (DAB) Kit (PV-6001, ZSGB Biotech, Inc., Beijing, China) and counterstained with hematoxylin. The average integrated optical density (IOD) of the positive cells was measured by ImageJ software.
A0277
Manufactured by Beyotime
Sourced in China
The A0277 is a laboratory equipment product. It serves as a core function in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
A0303, by Beyotime (6 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Normal goat serum (ngs), by OriGene (2 mentions)
Ab15580, by Abcam (1 mentions)
Ab215191, by Abcam (1 mentions)
Ab63929, by Abcam (1 mentions)
Af1817, by R&D Systems (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Bs 1571r, by Bioss Antibodies (1 mentions)
Goat serum, by OriGene (1 mentions)
Ab64238, by Abcam (1 mentions)
H2o2 solution, by Sinopharm (1 mentions)
Sl038, by Solarbio (1 mentions)
Hrp labeled goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
A11525, by ABclonal (1 mentions)
Inverted microscope, by Nikon (1 mentions)
A0428, by Beyotime (1 mentions)
Sab5700723, by Merck Group (1 mentions)
Pa5 27436, by Thermo Fisher Scientific (1 mentions)
13 protocols using a0277
1
Immunohistochemical analysis of Ki-67 expression
2
Immunoblotting for Kidney Injury Markers
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The kidney sections were ground using a homogenizer or the cells were scratched and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with proteinase inhibitor (Roche, Switzerland) for 30 min on ice. Protein concentration was quantified using the Bicinchoninic acid assay (Beyotime, Shanghai, China). Protein samples (50 μg) were used to perform immunoblotting as described previously42 (link). The specific primary antibodies used were: anti-GSDME (ab215191, Abcam, 1:1000), anti-caspase-3 (9662, CST, 1:1000), anti-cleaved caspase-3 (9661, CST, 1:1000), phospho-NF-κB p65 (3033, CST, 1:1000), NGAL (ab63929, Abcam, 1:1000), KIM-1 (AF1817, R&D systems, 1:1000), and anti-GAPDH (5174, CST, 1:10000). Peroxidase-conjugated goat anti-rabbit secondary antibodies (A0277, Beyotime, Shanghai, China) were used for immunoblotting.
3
Immunohistochemical Analysis of SNX5 Expression
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HCC tissues were cut into sections (thickness: 3 μm). The sections were deparaffinized with xylene and then hydrated. The sections were then heated in citrate buffer at 100°C for 1 min for antigen retrieval, and endogenous peroxidases were inactivated in 3% hydrogen peroxide for 10 min at 37°C. The sections were then incubated with primary anti-SNX5 antibody (ab250218, 1:100, Abcam, Shanghai, China) at 4°C overnight, followed by the incubation with secondary antibody (A0277, 1:1000, Beyotime, Shanghai, China) for 60 min at 37°C. Finally, 3,3-diaminobenzidine tetrahydrochloride was used to develop the color, and each section was scored by two independent pathologists. IHC score was calculated based on staining intensity score (0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining) and the proportion score (0, no staining; 1, 1–25% of the tumor cells were stained; 2, 26–50%; 3, 51–75%; and 4, more than 75% of the tumor cells were stained). IHC score = staining intensity score × proportion score. IHC score was used to evaluate the expression of SNX5: 0 points, negative; 1–6 points, weakly positive; 8–12 points, strongly positive.
4
Immunohistochemical Analysis of Retinal Tissues
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Retinal tissue sections (5-µm thick) were deparaffinized with xylene and gradient ethanol (100, 95, 85 and 75% ethanol). After antigen retrieval, the sections were treated with H2O2 and blocked with goat serum. Then, sections were incubated with primary antibodies against F-actin (1:200; bs-1571R; Bioss, Beijing, China) and GAP-43 (1:200; D163002; Sangon Biotech, Shanghai, China) at 4°C overnight and biotin-labeled secondary antibody (1:200; A0277; Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 30 min, followed by incubation with HRP-labeled avidin (A0303; Beyotime) at 37°C for 30 min. The sections were visualized with DAB, counterstained with hematoxylin and imaged under a microscope (Olympus Corp.).
5
Immunohistochemical Analysis of Retinal Tissue
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Retinal tissue sections (5 µm thick) were deparaffinized with xylene and an ethanol gradient (100%, 95%, 85%, and 75% ethanol). After antigen retrieval, the sections were treated with H2O2 and blocked with goat serum. Then, the respective primary antibodies were added to the sections in a volume of 100 µL and incubated overnight at 4°C. After primary incubation, biotin-labeled secondary antibody (1:200, A0277; Beyotime Biotechnology) was added and incubated at 37°C for 30 minutes, followed by incubation with horseradish peroxidase-labeled avidin (A0303; Beyotime) at 37°C for 30 minutes. The sections were visualized with DAB for color development and were counterstained with hematoxylin. Section images were captured using a microscope.
6
Immunohistochemical Analysis of Pancreatic Collagens
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Pancreatic tissue samples were fixed in 10% paraformaldehyde overnight at 4°C, embedded in paraffin and sectioned into 4-µm-thick slices. The slices were then incubated in an oven overnight at 55–65°C. After 30 min of deparaffinization in xylene, the slices were rehydrated in a graded ethanol series. The slices were then incubated in citrate buffer for 5 min at 25°C and treated in a microwave oven for 15 min for antigen repair at 65–75°C.
The sections were washed with 3% H2O2 and blocked with 10% goat serum (OriGene Technologies, Inc.) for 60 min at 37°C. The samples were incubated with the primary antibody against collagen type I (1:1,000; cat. no. 66761-1-Ig; ProteinTech Group, Inc.) and collagen type III (1:1,000; cat. no. 22734-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Species-specific secondary antibody (1:200; A0277; Beyotime Institute of Biotechnology) was added to the pancreatic tissue sections for 60 min at 37°C. Finally, the pancreatic tissue sections were stained with diaminobenzidine for 10 min at 25°C, and the cell nuclei were stained with hematoxylin for 3 min at 25°C. After sealing the sections with neutral resin, they were examined under a light microscope.
The sections were washed with 3% H2O2 and blocked with 10% goat serum (OriGene Technologies, Inc.) for 60 min at 37°C. The samples were incubated with the primary antibody against collagen type I (1:1,000; cat. no. 66761-1-Ig; ProteinTech Group, Inc.) and collagen type III (1:1,000; cat. no. 22734-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Species-specific secondary antibody (1:200; A0277; Beyotime Institute of Biotechnology) was added to the pancreatic tissue sections for 60 min at 37°C. Finally, the pancreatic tissue sections were stained with diaminobenzidine for 10 min at 25°C, and the cell nuclei were stained with hematoxylin for 3 min at 25°C. After sealing the sections with neutral resin, they were examined under a light microscope.
7
Immunohistochemical Staining Protocol
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Paraffin sections were de-waxed and re-hydrated, followed by adding 3% H2O2 solution (10011218, Sinopharm, China) to incubate 15min at room temperature to block endogenous peroxidase activity. Sections were placed in citrate buffer, and microwave was used to repair antigen. 5% normal goat serum (SL038, Solarbio, China)
was added for 15min blocking at room temperature. LIS1 antibodies, Ki67 antibodies (27309-1-AP, Proteintech, China) or CK-19 antibodies (1:100, ThermoFisher, USA) were added to maintain overnight at 4 °C. Horseradish peroxidase labeled rabbit secondary antibody (1:200, A0277, Beyotime, China) was added for 30min incubation at 37 °C. Horseradish enzyme labeled streptavidin working solution (A0303, Beyotime, China) was dripped for 30 min incubation at 37 °C. After DAB color development (ab64238, Abcam, UK), hematoxylinwas was used to stain sections at room temperature.
was added for 15min blocking at room temperature. LIS1 antibodies, Ki67 antibodies (27309-1-AP, Proteintech, China) or CK-19 antibodies (1:100, ThermoFisher, USA) were added to maintain overnight at 4 °C. Horseradish peroxidase labeled rabbit secondary antibody (1:200, A0277, Beyotime, China) was added for 30min incubation at 37 °C. Horseradish enzyme labeled streptavidin working solution (A0303, Beyotime, China) was dripped for 30 min incubation at 37 °C. After DAB color development (ab64238, Abcam, UK), hematoxylinwas was used to stain sections at room temperature.
8
Ovarian Tissue Immunohistochemistry Analysis
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The ovarian tissue slices as above mentioned was utilized to immunohistochemistry examination. Sections were incubated with primary antibodies against α-SMA (55135-1-AP, Proteintech, Wuhan, China) or CD31 (A11525, Abclonal, Wuhan, China) overnight at 4°C. After washing in PBS, slides stained with α-SMA were incubated with goat against rabbit biotin-labeled antibody (A0277, Beyotime, Shanghai, China) in PBS solution for 60 min at 37°C and conjugated with HRP-labeled Streptavidin (A0303, Beyotime) for 30 min at room temperature. The HRP-labeled goat anti-rabbit antibody (#31460, Thermo Fisher Scientific) was prepared to conjugate with CD31. Then slides were colorated using DAB reagent and counterstained with hematoxylin. The images were shot at × 100 or ×400 magnification.
9
Immunohistochemical Analysis of Lung Tissue
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Firstly, the lung sections were were cut into 5 μm using microtome, deparaffinized and rehydrated. With incubation at 3% H 2 O 2 at 37 °C for 10 min, the endogenous peroxidase activity was eliminated. The lung sections were boiled in antigen retrieval buffer containing citratehydrochloric acid for 15 min and blocked with 5% normal goat serum (OriGENE Technologies, Inc.) for 30 min. Then the sections were incubated with anti-MPO antibody (22225-1-AP, proteintech) or anti-CD68 antibody (28058-1-AP, proteintech) overnight at 4 °C. The next day, the sections were incubated with biotin-labeled secondary antibody working solution (1:200, A0277, Beyotime, Shanghai, China; Goat anti-rabbit IgG-HRP) at 37 °C for 30 min. The morphology of lung slides was scanned with Nikon inverted microscope (Nikon, Tokyo, Japan).
10
Examining NLRP3 and Microglia in ARDS
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At 24 h after ARDS induction, three rats from each group were deeply anesthetized with pentobarbital sodium (40 mg/kg), followed by transcardial perfusion with 100 mL PBS and 300 mL 4% paraformaldehyde (PFA; C104190; Aladdin, Shanghai, China). The hippocampal and cortex tissues were fixed in 4% PFA for 12 h and embedded in optimal cutting temperature compound (Sakura, CA, USA). Hippocampal tissue slices (50 μm) were renatured in citrate buffer (pH 6.0) at 108°C for 5 min. For immunohistochemistry (IHC), the renatured slices were pretreated with 1% H2O2 at room temperature (RT) for 15 min. After three PBS washes, the slices were masked with 10% NGS for 1 h in PBS. Slices were incubated with anti‐NLRP3 (SAB5700723; Sigma‐Aldrich) at 4°C for 24 h, followed by incubation with biotin‐labeled goat antibody against rabbit immunoglobulin G (IgG; 1:250; A0277; Beyotime) for 2 h and avidin–biotin complex for 1 h at RT. After three PBS washes, the slices were immersed in 3,3′‐diaminobenzidine solution at 37°C for 10 min.
For immunofluorescence, after masking with NGS, the slices were probed with an antibody against Iba‐1 (1:1000; PA5‐27436; Invitrogen, CA, USA) at 4°C for 24 h, followed by incubation with Alexa 488‐conjugated secondary antibody against rabbit IgG (1:250; A0428; Beyotime). Tissue slices were stained with DAPI before sealing.
For immunofluorescence, after masking with NGS, the slices were probed with an antibody against Iba‐1 (1:1000; PA5‐27436; Invitrogen, CA, USA) at 4°C for 24 h, followed by incubation with Alexa 488‐conjugated secondary antibody against rabbit IgG (1:250; A0428; Beyotime). Tissue slices were stained with DAPI before sealing.
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