Paraffin‐embedded renal tissues were cut into 5‐µm‐thick slices. The sections were incubated with the primary antibody rabbit anti‐human laminin beta‐2 (1:50; 223869, Life Science) at 4°C overnight and then incubated with secondary antibody conjugated (PV‐9000, Zhongshan Golden Bridge Biotechnology, China) to horseradish peroxidase. After counterstaining with haematoxylin, the sections were photographed under a BX53 microscope (Olympus).
Pv 9000
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
The PV-9000 is a high-performance laboratory equipment designed for precise measurements and analysis. It features advanced technology to deliver accurate and reliable results. The core function of the PV-9000 is to provide precise measurements and data analysis capabilities for scientific research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab39012, by Abcam (2 mentions)
Ab34712, by Abcam (2 mentions)
Bx53 microscope, by Olympus (1 mentions)
Goat serum, by Agilent Technologies (1 mentions)
Leica x software, by Leica camera (1 mentions)
Leica dmi8 microscope, by Leica camera (1 mentions)
3 3 diaminobenzidine (dab), by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab78237, by Abcam (1 mentions)
Ab108403, by Abcam (1 mentions)
Pv 9000 two step immunohistochemical kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
H600l microscope and image analysis system, by Nikon (1 mentions)
Ab96869, by Abcam (1 mentions)
Zli 9022, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab52915, by Abcam (1 mentions)
Ab28482, by Abcam (1 mentions)
Diaminobenzidine dab kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Sc 28887, by Santa Cruz Biotechnology (1 mentions)
Pv 9003, by Zhongshan Golden Bridge Biotechnology (1 mentions)
16 protocols using pv 9000
1
Immunohistochemical Analysis of Renal Laminin
2
Immunohistochemical Analysis of Cytoskeletal Proteins
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Morphology was examined in tissue sections (5 μm) stained with H&E. The expression of FN, calpain-2, E-cadherin, and vimentin was analyzed using a two-step Immunohistochemical Stain Detection System (PV-9000, Zhongshan Golden Bridge Biotechnology) according to the manufacturer’s instructions. Briefly, the sections were deparaffinized, and antigen retrieval was achieved by microwave heating in 0.01 M citrate buffer for 20 min. The sections were sequentially incubated with 3% H2O2, blocked with goat serum (Dako) for 30 min, and incubated with the following primary antibodies at 4 °C overnight: FN (1:300), E-cadherin (1:250), calpain-2 (1:250), and vimentin (1:350). The sections were successively incubated with biotinylated secondary antibodies for 20 min and streptavidin-HRP for 2 min, and then stained with DAB substrate and counterstained with hematoxylin. Results were captured using a Leica DMI8 microscope and Leica X software (Leica) at ×400 magnification.
3
Immunohistochemical Detection of SOX2
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The SOX2 antibody was purchased from Bethyl Laboratories, Inc. (A301-741A; Montgomery, TX, USA). Immunohistochemical reagent (PV 9000 and DAB) coloring solution was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Tissue sections were dewaxed in xylene, hydrated in gradient ethanol (100, 95, 90, 85, 80 and 75%), placed in 3% hydrogen peroxide and incubated for 10 min to block endogenous peroxidase. Subsequently, the sections were rinsed with phosphate-buffered saline 3 times, and incubated with 5% goat serum at 25°C for 30 min, followed by incubation with rabbit polyclonal SOX2 primary antibody (Abcam, Cambridge, MA, USA; catalog no. ab97959; dilution of 1:500) at 4°C overnight. The following day, the sections were incubated with PV 9000 at room temperature, and color was developed using DAB solution. After rinsing the slides in deionized water, the sections were counterstained with hematoxylin and decolorized with 1% hydrochloric acid, returned to blue following 1% ammonia and dehydrated with gradient ethanol before sealing.
4
Immunohistochemistry of Colorectal Cancer Tissue
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The immunohistochemistry process was conducted by pathologists in the Department of Pathology of the two Hospitals. The paraffin-embedded CRC tissue was cut into 4-μm thickness sections. The paraffin sections were performed immunohistochemistry detection successively as dewaxed, antigen retrieval, block endogenous peroxidase, serum blocking, primary antibodies incubation, horseradish peroxidase (HRP)-conjugated secondary antibody incubation, 3,3N-Diaminobenzidine staining, counterstained by haematoxylin, graded dehydration and mounting as previous described (Zeng et al., 2018 (link)). The primary antibodies for MMR proteins were ready-to-use antibodies: MLH1 (ZM-0154, Zhongshan Goldenbridge Biotechnology Beijing, P.R. China), MSH2 (ZA-0622, Zhongshan Goldenbridge Biotechnology), MSH6 (ZA-0541, Zhongshan Goldenbridge Biotechnology) and PMS2 (ZA-0542, Zhongshan Goldenbridge Biotechnology). The polymer HRP detection system (PV-9000, Zhongshan Goldenbridge Biotechnology) was used to detect the antigen–antibody binding reaction according to the manufacturer’s protocol (Zeng et al., 2018 (link)). The staining scoring criterion of the grading of staining intensity and the percentage of positive cells was adopted as previously mentioned (Sun et al., 2014 (link)). The results were evaluated by two independent pathologists with anonymous patient’s information.
5
Immunohistochemical Analysis of CD20, CD38, and AOC1
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The expression of CD20, CD38, and AOC1 in adjacent cancer tissue sections were explored. After fixed in paraformaldehyde for 15 min, the sections were immersed in 0.25% Triton X-100 (ZLI-9308, Zhongshan Goldenbridge Biotechnology Ltd. Co., Beijing, China) for 15 min and blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) for 30 min. Then, the sections were incubated with antibodies against CD20 (Abcam; ab78237; 1:100), CD38 (Abcam; ab108403; 1:500), and AOC1 (ABP1, Abcam; ab278497; 1:500) at 4 °C overnight, followed by rewarming at room temperature for 30 min. Then, PV-9000 two-step immunohistochemical kit was used according to the manufacturer’s instructions (PV-9000, Zhongshan Goldenbridge Biotechnology Ltd. Co., Beijing, China), and a DAB kit was used subsequently to detect antigen-antibody binding.
6
Collagen II and MMP-13 Immunohistochemistry
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In addition to histomorphological evaluation, serial sections were stained for assessment of collagen II and MMP‐13 contents. After deparaffinization and rehydration of the tissue sections, endogenous peroxidase activity was blocked by 3% H2O2 for 20 min. The proteins were immunostained using a 2‐step method according to the kit manufacturer's instructions. The sections were incubated with rabbit polyclonal anti‐collagen II antibody (ab34712, 1:100; Abcam, Cambridge, MA), and rabbit polyclonal anti‐MMP‐13 antibody (ab39012, 1:50; Abcam) overnight at 4°C. The slides were washed three times in PBS followed by incubation for 20 min at 37°C with an anti‐mouse/rabbit immunoglobulin G (IgG) detection system (PV‐9000; Zhongshan Goldenbridge Biotechnology Co., China) and visualized with diaminobenzidine. Nuclei were counterstained with hematoxylin for 5 min. The optical densities of the stained slides were measured using image analysis software (Nikon H600L Microscope and image analysis system, Japan). Collagen II was expressed by relative intensity. MMP‐13 was expressed by the percentage of positive cells.
7
Immunohistochemical Analysis of Mesothelin
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Paraffin-embedded MPM specimens were cut into 5 mm sections for immunohistochemistry analysis. Sections were deparaffinized in xylene and then rehydrated with ethanol. For antigen retrieval, the specimens were incubated in a microwave oven (95 °C) in ethylene diamine tetraacetic acid (EDTA) (ZLI-9068, Zhongshan Golden Bridge Bio-technology, China) for 15 min. After natural cooling, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min at room temperature. Then, sections were washed with phosphate buffer saline (PBS) for 5 min. Nonspecific antibody binding was blocked with goat serum (ZLI-9022, Zhongshan Golden Bridge Biotechnology, China) for 10 min. Subsequently, the specimens were incubated with rabbit polyclonal anti-mesothelin antibody (1:200, ab96869, abcam, UK) at 4 °C for overnight. Rewarming at 37 °C and then washed with PBS, the specimens were incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (PV-9000, Zhongshan Golden Bridge Bio-technology, China). Counter-stained with hematoxylin, the sections were rinsed, dehydrated and covered.
8
Immunohistochemical Evaluation of Cartilage Markers
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In addition to the histomorphological evaluation, serial sections were stained for COL2A1, MMP-3, and MMP-13. After deparaffinization and rehydration of the tissue sections, the proteins were immunostained using a two-step method following the manufacturer’s instructions for the kit. The sections were incubated with rabbit polyclonal anti-COL2A1 antibody (ab34712, 1:50; Abcam, Cambridge, MA, United States), rabbit polyclonal anti-MMP-3 antibody (ab52915, 1:50; Abcam), and rabbit polyclonal anti-MMP-13 antibody (ab39012, 1:50; Abcam) overnight at 4°C. The slides were washed three times in PBS followed by a 20 min incubation at 37°C with an anti-mouse/rabbit IgG detection system (PV-9000, Zhongshan Goldenbridge Biotechnology Co., Beijing, China) and visualized with diaminobenzidine. Nuclei were counterstained with hematoxylin for 5 min. Negative control sections were prepared using the same protocol, but the primary antibody was replaced with PBS. The optical density of the stained slides was measured using image analysis software (NikonH600L microscope and image analysis system, Tokyo, Japan). COL2A1 and MMP-3 were expressed as relative intensities. MMP-13 was expressed as a percentage of positive cells.
9
Immunohistochemical Detection of SREBP-2 in Liver
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Immunohistochemistry was performed using a universal two-step detection kit (PV9000, Zhongshan Golden Bridge Biotechnology Co, Beijing, China) to detect SREBP-2 protein expression in accordance with the manufacturer's instructions. Briefly, the liver sections were dewaxed, repaired antigen retrieval, and incubated with endogenous peroxide blocker for 10 minutes at room temperature and then incubated with the primary antibody (1:100, abcam, Burlingame, CA, USA, ab28482) overnight at 4°C. The protein expression was observed by laser-scanning microscopy (Zeiss Vert.A1, Carl Zeiss Canada).
10
Immunohistochemical Analysis of PHB1 in Prostate Cancer
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The cancer-adjacent normal tissues and PCa specimens were collected from patients undergoing radical prostatectomy at Qilu Hospital, Shandong University (Jinan, China) and then were made into tissue microarray (TMA). TMA slides staining with H&E were reviewed by two pathologists according to the WHO histologic classification of PCa. After that, the IHC staining was performed with General-purpose two-step immunohistochemical detection kit (PV-9000, Zhongshan Golden Bridge Biotechnology Co, Beijing, China). For PHB1 expression in tissues, the staining intensity was scored from 1 to 3: 1, weak; 2, moderate; 3, strong. The information of antibody used is summarized in Supplementary Table S2 .
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