Immunohistochemistry was performed on 5 micron-thick, formalin fixed, paraffin-embedded tumor sections, which were initially deparaffinized, rehydrated and heated in citric acid (pH 6.0) for 20 min in a microwave oven. After antigen retrieval, endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min, followed by 30 min blocking incubation in goat serum (Histostain™-Plus Kits; ZSGB-BIO). The slides were then incubated overnight at 4°C with primary antibody against USP33. Next, the slides were incubated with biotinylated antibody (Histostain™-Plus Kits; ZSGB-BIO) for 30 min at room temperature. Finally, slides were visualized by 3,3-diaminobenzidine (DAB) staining. Stained slides were digitalized with Scanscope XT (Aperio Technologies, Vista, CA). To quantitate the state of USP33 protein expression, the mean percentage of positive tumour cells was determined in at least five random fields at 200× magnification in each section. The intensity of the USP33-immunoreaction was scored as follows: 0, negative; 1+, weak; 2+, moderate; and 3+, intense. The percentage of positive tumour cells and the staining intensity then were multiplied to produce the USP33-immunohistochemical staining score.
Histostain plus kit
Manufactured by ZSGB-BIO
Sourced in China
The Histostain-Plus Kit is a laboratory equipment designed for immunohistochemical (IHC) staining procedures. It provides the necessary reagents and components for the detection and visualization of target antigens in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Dab kit, by ZSGB-BIO (4 mentions)
Dab horseradish peroxidase color development kit, by ZSGB-BIO (2 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by ZSGB-BIO (2 mentions)
Microscopy digital camera system, by Olympus (2 mentions)
Il 1β, by Cell Signaling Technology (2 mentions)
Goat anti rabbit secondary antibody, by ZSGB-BIO (2 mentions)
Trypsin edta, by Solarbio (2 mentions)
Mouse anti vim, by Abcam (1 mentions)
Rabbit anti α sma, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Nichirei Biosciences (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Ab90088, by Abcam (1 mentions)
Ab128698, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Ifn γ antibody, by Abcam (1 mentions)
Bx63 microscope, by Olympus (1 mentions)
Anti acadm, by Abcam (1 mentions)
Anti acaa1, by Proteintech (1 mentions)
29 protocols using histostain plus kit
1
Quantitative Immunohistochemical Analysis of USP33
2
Immunohistochemical Profiling of Liver Fibrosis
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Mouse liver tissues were formalin fixed, embedded in paraffin and sectioned at 4 μm. Slides were routinely processed for immunohistochemically stained using the following antibodies: rabbit anti-αSMA (1:200, Abcam, MA, USA), rabbit anti- FLNA (1:1000, Abcam, MA, USA), mouse anti-VIM (1:500, Abcam, MA, USA). Visualization was perfomed using the Histostain-Plus Kit (ZSGB-BIO). For immunofluorescence studies, the isolated HSCs were cultured in DMEM/F12 supplemented with 10% fetal bovine serum for 1 month and kept stable proliferation rate and growth characteristics. These primary activated HSCs grown on cover slips were fixed and stained using the above antibodies. 20x pictures for each slip was analyzed using Image J software. The detail of mice liver fibrosis model and isolation of mice HSCs are provided in the supporting information .
3
Immunohistochemical Evaluation of Cartilage Markers
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Paraffin sections (4 μm) were prepared, and IHC was performed with a Histostain-Plus kit (ZSGB-BIO, Beijing, China). The primary antibodies included anti-COL2A1, COL10A1, RUNX2, ID1, and p-SMAD1S463/465 antibodies. A DAB Horseradish Peroxidase Color Development Kit (ZSGB-BIO) was used for detection. Immunostaining evaluations were performed independently by experimenters blinded to sample identity. The staining intensity was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percent of positivity was also scored according to 5 categories: 0 (<5%), 1 (5%−25%), 2 (25%−50%), 3 (50%−75%), and 4 (>75%). Then, the value of the percent positivity score was multiplied by the staining intensity score to generate final expression scores, which ranged from 0 to 12.
4
Immunostaining of Gamma-Glutamyl Transferase in Mice
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Male C57BL/6 mice (n=4; age, four weeks; weight, 20–30 g) were purchased from the experimental animal center of the Second Affiliated Hospital of Harbin Medical University. The animal experiments were approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University. The mice were anesthetized with 10% chloral hydrate (250 mg/kg; Wuhan Hechang Chemical Co., Ltd., Wuhan, China) by intraperitoneal injection and perfused transcardially with 4% paraformaldehyde (Tianjin Guangfu Chemical Co., Ltd., Tianjin, China) in phosphate-buffered saline (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The mandibles were removed and immersed in 4% paraformaldehyde at 4°C for 48 h. The tissues were subsequently decalcified and embedded in paraffin (Shanghai Hushi Laboratorial Equipment Co., Ltd, Shanghai, China). Five-micron-thick sections were analyzed by immunohistochemistry using a mouse monoclonal antibody against gamma-glutamyl transferase (1:400 dilution; cat. no. ab55138; Abcam, Cambridge, MA, USA). The sections were then incubated with a biotinylated secondary antibody using a Histostain-Plus kit (ZSGB-BIO, Beijing, China). The color reaction was finally developed using 3,3′-diaminobenzidine (Nichirei, Tokyo, Japan). The sections were observed using a light microscope (Eclipse 80i; Nikon, Tokyo, Japan).
5
Immunohistochemical Analysis of PLOD and Collagen I
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Tissue sections were dewaxed and hydrated, then immunohistochemical staining was performed using the Histostain-Plus Kit (PV-9000, ZSGB-BIO, China; 20—40 s), followed by a DAB Kit (ZLI-9017, ZSGB-BIO, China; 1–10 min). The sections were incubated with an anti-PLOD1 antibody (LH1, 1: 100, Abcam, ab171140), anti-PLOD2 antibody (LH2, 1: 100, Abcam, ab90088), anti-PLOD3 antibody (LH3, 1: 100, Abcam, ab128698), or an anti-collagen I antibody (COLI, 1: 100, Abcam, ab6308) overnight at 4°C (12–16 h). This was followed by incubation with conjugated secondary antibodies at room temperature for 1 h. DAB and hematoxylin were deconvoluted using Image J software, and images were pseudocolored to assess their colocalization. Samples with yellow granular staining were considered to have positive expression of the protein.
6
Developmental Mouse Tissue Analysis
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Tissues from E14.5, E16.5, and P0 mice were dissected and fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin. Sections for histological analysis were rehydrated and stained with hematoxylin‐eosin. Immunofluorescence was performed with Histostain‐Plus Kit (ZSGB‐BIO, Catalog No. SPN‐9002). Primary antibodies included: 1:400 dilution of anti‐myosin antibody (Sigma, Catalog No. M4276); 1:200 dilution of anti‐Ki67 antibody (Abcam, Catalog No. ab16667). Detection was conducted using 1:1,000 dilution of anti‐mouse IgG fragment, Alexa Fluor 555 conjugate (CST, Catalog No. #4409), and 1:1,000 dilution of anti‐rabbit fragment, Alexa Fluor 555 conjugate (CST, Catalog No. #4413S). Nucleus was stained by DAPI in a final concentration of 0.1 μg/ml (CST, Catalog No. #4083). TUNEL assay was performed according to the manufacturer's instructions (MBL, Catalog No. 8445). Quantification of Ki67‐positive rate and TUNEL‐positive rate was conducted using ImageJ (version 1.51) software.
7
Immunohistochemical analysis of cartilage and joint
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Paraffin sections of the cartilage pellets and mouse knee joints were prepared for IHC experiments using the Histostain‐Plus Kit (ZSGB‐BIO, China). The antibodies included anti‐COL2A1 (1:200), anti‐SOX9 (1:500), anti‐MMP9 (1:200) and anti‐MMP13 (1:1000) antibodies. Detection was performed using a DAB Horseradish Peroxidase Color Development Kit (ZSGB‐BIO, China), and staining intensity was scored according to the previously published article.24
8
Tissue Deparaffinization and Antigen Retrieval
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The sections of liver tissues were deparaffinized by incubation at 60°C for 24 h, followed by two successive immersions in xylene at 56°C for 30 min each, followed by hydration in solutions with decreasing concentrations of ethanol (100%, 95%, 80%, and 70%). For antigen retrieval, the slides were incubated in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 30 min, after preheating for 14 min. Endogenous peroxidase activity was blocked by incubating with a 3% peroxide/methanol solution for 15 min at room temperature. Other steps were performed according to the protocol of a commercially available Histostain–plus kit (ZSGB-BIO, Beijing, China).
9
Immunohistochemical Detection of Hepatitis E Virus
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Livers, lungs, kidneys, spleens, lymph nodes, duodenums, jejunums, ileums, SR, and appendices were collected and fixed in 4% paraformaldehyde for 48 h. The fixed tissues were then processed routinely in paraffin and 4-mm sections were prepared. Monoclonal mouse anti-HEV ORF2 antibody (1∶300 dilution; Beijing Protein Institute, Beijing, China) was used as the primary antibody. The primary antibody was added to the sections and incubated at 37°C for 2 h. Immunohistochemical staining was performed according to the instructions of the Histostain™-Plus Kit (ZSGB-BIO, Beijing, China). 3,3′- Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Beijing, China) was applied for 10 min to visualize the antigen–antibody reaction, and then Gill's hematoxylin was applied as the background stain. The primary antibody was replaced with phosphate-buffered saline in the negative control. The slides were then observed under an Olympus microscope (Japan).
10
Immunohistochemical Staining of RegIIIγ and IFNγ
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Tissue slides were incubated with the corresponding primary RegIIIγ antibody (Abcam, Cambridge, England) or IFNγ antibody (Abcam) and then incubated with HRP-conjugated antibody against rabbit or mouse IgG using a Histostain-Plus Kit (ZSGB-BIO, Beijing, China). The slides were counterstained with hematoxylin and finally inspected under a microscope (Leica, Wetzlar, Germany).
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