The human NSCLC tissue microarrays and xenograft tumor tissue sections were deparaffinized in xylene and rehydrated through an ethanol series, followed by microwave in citrate buffer (pH 6.0) to repair antigens and treatment with 3% hydrogen peroxide to inhibit endogenous peroxide activities. These tissue microarrays and sections were then blocked with 5% normal goat serum for 20 min at room temperature, and treated with antibody against PDIA6 (1:100; 18233–1-AP; RRID: AB_10805765), Ki-67 (1:800; 27309–1-AP; RRID: AB_2756525), MAP4K1 (1:100; bs-4134R; RRID: AB_11120767), or phospho-MAP4K1 (1:100; bs-5494R; RRID: AB_11093913) at 4 °C overnight. After washing, tissue microarrays and sections were incubated with the pv-9000 kit (ZSGB-Bio, Beijing, China), visualized with 3,3-diaminobenzidine solution (ZSGB-Bio) treatment and counterstained with hematoxylin. The percentage (%) of positively stained cells was classified as 1 (0%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (76%–100%), while the staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). We then multiplied these two scores to reach a staining index. The staining index <6 was defined as low PDIA6 expression, while the staining index ≥6 as high PDIA6 expression.
Pv 9000 kit
Manufactured by ZSGB-BIO
Sourced in China
The PV-9000 kit is a laboratory equipment designed for scientific research and analysis. It is a comprehensive package that includes various components necessary for conducting experiments and studies. The core function of the PV-9000 kit is to provide researchers with the tools and resources required to perform a range of scientific tasks efficiently and accurately.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine solution, by ZSGB-BIO (1 mentions)
Dh0006 2, by Leagene (1 mentions)
Cm1900, by Leica (1 mentions)
Sodium citrate buffer, by Wuhan Servicebio Technology (1 mentions)
Pannoramic viewer, by 3DHISTECH (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Diaminobenzidine dab, by Beyotime (1 mentions)
Hematoxylin, by Absin (1 mentions)
Zli 9067, by ZSGB-BIO (1 mentions)
Za 0502, by ZSGB-BIO (1 mentions)
Zm 0069, by ZSGB-BIO (1 mentions)
Za 0550, by ZSGB-BIO (1 mentions)
Eclipse 50i microscope, by Nikon (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
P rip3, by Abcam (1 mentions)
Anti tumour necrosis factor α tnf α, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
11 protocols using pv 9000 kit
1
Quantitative Analysis of PDIA6, Ki-67, and MAP4K1 in NSCLC
2
Histological Analysis of Tumorous Nude Mouse Skulls
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The whole skull of each tumorous nude mouse was collected, fixed in 4% paraformaldehyde for 24–48 h, embedded in paraffin, cut into continuous 4-µm thick sections, and stained with hematoxylin and eosin (LEAGENE, DH0006-2 Beijing, China). Immunohistochemical staining was performed using a ZSGB-BIO PV-9000 kit (Beijing, China) according to the manufacturer’s instructions. Tissue sections from paraffin-embedded human GBM specimens and xenograft tissues were stained with specific antibodies (refer to Supplementary information) or nonspecific IgG as negative controls.
3
Immunohistochemical Staining of ANXA3 in Rat CNS
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Immunohistochemical staining was performed using a PV-9000 Kit (ZSBiO, China) according to the product instructions [17 (link)]. Briefly, brain and spinal cord sections from rats were sliced into 10 μm thick sections (Leica CM1900), which were then subjected to antigen retrieval in a heated citrate buffer (pH 6.0) and incubated with 3% H2O2 for 10 min at room temperature to block endogenous peroxidase activity. Rabbit anti-ANXA3 antibody (1 : 1000, Sigma, USA) was used as a primary antibody. After the sections were incubated with peroxidase-labeled goat anti-rabbit/mouse IgG antibody, diaminobenzidine (DAB) was used as a chromogen.
4
Immunohistochemical Staining Protocol
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The sections were baked in a 60°C oven for 2 h and then dewaxed with gradient ethanol. For antigen retrieval, tissue sections were placed in sodium citrate buffer (Servicebio Technology, Wuhan, China) and boiled for 18 min. Then, a PV-9000 kit (ZSGB-BIO, Beijing, China) was used according to the manufacturer’s instructions. The tissue sections were incubated with primary antibodies overnight at 4°C. Finally, DAB staining, hematoxylin redyeing and hydrochloric acid alcohol differentiation were performed. The images were captured with an Pannoramic Viewer (3DHISTECH, RRID: SCR_014424 ).
5
ITGB1 Protein Expression in Cervical Cancer
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The protein abundance of ITGB1 in CC tissues and normal cervical tissues was detected using IHC. Primary antibody against ITGB1 were purchased from ImmunoWay Biotechnology (SuZhou, China). Tissue samples were fixed with 4% paraformaldehyde and processed using standard procedures of dehydration, fixation, embedding, and slicing. The slides were then treated with primary antibodies at 4°C overnight. Prior to incubating with the secondary antibody, the slides were washed three times with PBS. The PV-9000 Kit (Zsbio, Beijing, China) was used as the secondary antibody, and the slides were incubated for 1 hour at room temperature, protected from light. The substrate diaminobenzidine (DAB, Beyotime) was used for antibody detection, and the slides were counterstained with hematoxylin (Beyotime). The cell nucleus was stained blue by hematoxylin. The IHC images were obtained using a Zeiss light microscope and analyzed for mean optical density using Image J software.
6
Immunohistochemical Analysis of CD31, S100A9, and TXNDC12
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We collected paraffin sections of patients with non-cancer or cancer from Hunan Cancer Hospital and performed immunohistochemical staining. Then Samples were dewaxed with ethanol and blocked to inhibit endogenous peroxidase activity. They were retrieved antigen by autoclave boiling for 20min. Samples were incubated overnight at 4°C with rabbit anti-CD31 (ZEN-BIOSCIENCE, Chengdu, China, 1:100), anti-S100A9 (Abclonal, Wuhan, China, 1:100), anti-TXNDC12 (Abclonal, Wuhan, China, 1:100), followed by incubation with the secondary antibody PV-9000 Kit (zsbio, Beijing, China) at 37°C for 20 min. Cell nuclei were stained blue with use of hematoxylin. The ImageJ Software 1.53 (United States) was used to analyze protein expressions and perform statistics.
7
Immunohistochemical Staining of FFPE Tumor Tissues
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Formalin-fixed, paraffin-embedded (FFPE) blocks of tumor tissue were sliced into 5 µm sections, deparaffinized in xylene (3×5 minutes) and then rehydrated through graded concentrations of ethanol (2×2 minutes in 100% ethanol, 1×2 minute in 95% ethanol, 1×2 minute in 75% ethanol). Tissue sections were then heated in EDTA solution (ZLI-9067; ZSGB-BIO, Beijing, China), for 150 seconds. After cooling, the sections were washed with PBS and then incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit antibodies against CD34 (ZA-0550; ZSGB-BIO), CD99 (ZA-0577; ZSGB-BIO), Bcl-2 (ZA-0536; ZSGB-BIO), Ki67 (ZA-0502; ZSGB-BIO), and S100 (ZA-0225; ZSGB-BIO), as well as mouse antibodies against CK5/6 (ZM-0313; ZSGB-BIO) and CK-pan (ZM-0069; ZSGB-BIO). Slides were washed again and incubated with secondary antibodies using a PV-9000 kit (ZSGB-BIO) according to the manufacturer’s manual. The slides were then stained with hematoxylin (Absin, Fuzhou, China).
8
Immunohistochemical Analysis of Tumor Tissue
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The embedded tumor tissue was sectioned into 5 μm slices. After heating and antigen retrieval (incubating the slices at 98 °C in Tris-EDTA buffer for 5 min), tissue sections were subjected to IHC analysis according to standard protocol34 (link). Primary antibodies (Table S1 ) and secondary goat anti-mouse or anti-rabbit antibody conjugated with horseradish peroxidase (PV-9000 Kit, ZSGB-BIO) were used. The negative controls were made by omitting the primary antibodies for each staining. Target proteins were visualized with diaminobenzidine and images were obtained with a Nikon ECLIPSE 50i microscope.
9
Protein Expression and Localization in Brain Tissue
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Total proteins were harvested from the cortex and hippocampus. The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam), anti‐p‐MLKL (Abcam), anti‐tumour necrosis factor‐α (TNF‐α) (Abcam), anti‐IL‐1β (Abcam), anti‐IL‐6 (Abcam) and anti‐β‐actin (Cell Signaling Technology). After incubation with secondary antibodies (Solarbio), the positive bands were visualized using an ECL substrate (Solarbio). For immunohistochemical analysis, brain tissue sections were incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam) and anti‐p‐MLKL (Abcam). Subsequently, a PV9000 kit (ZSGB‐BIO) was used for follow‐up. For immunofluorescence analysis, brain tissues were blocked with goat serum for 1 hour and incubated with primary antibodies against p‐RIP3 (Abcam), p‐MLKL (Abcam), NeuN (Abcam), Iba‐1 (Wako; Servicebio) and GFAP (Servicebio) overnight at 4℃. Then, the tissues were incubated with Alexa Fluor® 594 goat anti‐rabbit IgG and Alexa Fluor® 488 goat anti‐mouse IgG secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific).
10
Immunohistochemical Analysis of Cervical Tissue
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We collected normal cervical tissue and cervical cancer tissue at the Hunan Provincial Cancer Hospital for immunohistochemical staining, and it has been reviewed and approved by the Ethics Committee of Hunan Cancer Hospital. After dewaxing of sections, heat antigen retrieval was performed. The primary antibodies SFT2D1 (Immunoway, USA, 1:100) and CD31 (ZenBio, China, 1:100) were incubated overnight at 4 °C. The secondary antibodies were incubated for 20 min using the PV-9000 kit (ZSGB-BIO, China). DAB reagent (ZSGB-BIO, China) was used for antibody staining, with brown-yellow indicating positive signal areas. Cell nuclei were stained blue with hematoxylin (Servicebio, China). Images were captured using microscope (Zeiss, Germany) and analyzed using Image J software (1.53, USA).
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