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Sp 9000 histostain plus kit

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The SP-9000 Histostain-Plus kits are laboratory equipment designed for immunohistochemistry (IHC) staining procedures. These kits provide the necessary reagents and components to perform IHC staining on tissue samples. The core function of the SP-9000 Histostain-Plus kits is to enable the visualization of specific target proteins or antigens within tissue sections.

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Lab products found in correlation

Image pro plus software, by Media Cybernetics (2 mentions) Adamts5, by Abcam (2 mentions) Digital pathology slide scanner, by 3DHISTECH (1 mentions) Pakt308, by Cell Signaling Technology (1 mentions) Runx2, by Santa Cruz Biotechnology (1 mentions) Aggrecan, by Merck Group (1 mentions) Mmp13, by Proteintech (1 mentions) P creb, by Abcam (1 mentions) Cleaved caspase 3, by Boster Bio (1 mentions) P smad3, by Cell Signaling Technology (1 mentions) Ab39012, by Abcam (1 mentions) Ab54210, by Abcam (1 mentions) Image pro plus version 5, by Media Cybernetics (1 mentions) Ab34712, by Abcam (1 mentions) Ab18256, by Abcam (1 mentions) Anti hif 2α, by Abcam (1 mentions) Anti osteocalcin, by Santa Cruz Biotechnology (1 mentions) Anti vhl, by Abcam (1 mentions) Anti runx2, by Santa Cruz Biotechnology (1 mentions) Anti pdk1, by Abcam (1 mentions)

10 protocols using sp 9000 histostain plus kit

1

SP and LiCl Induced Cell Differentiation

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The isolated cells were cultured in a 6-well plate (104 cells/well) for 24 h. The cells were treated with 10-7 M SP or LiCl, a Wnt agonist, to induce differentiation for 12 days. The cells were rinsed five times with PBS, fixed with 4% paraformaldehyde solution for 15 min at room temperature, and permeated with 0.5% Triton X-100-PBS solution for 5 min. The cells were incubated with primary antibodies to anti-cytokeratin 18 antibody (CK18) (rabbit polyclonal antibody, 1:50, Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequent procedures were performed according to the SP-9000 Histostain-Plus Kit instructions (ZSGB-BIO, Beijing, China). The cells were observed under a microscope (Bio-Rad, Hercules, CA, USA).
Cheng P., Sun X., Yin D., Xu F., Yang K., Qin L., Dong Y., Guo F., Chen A., Zhang W, & Huang H. (2015). Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation. Cell & Bioscience, 5, 5.
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2

Immunohistochemical Analysis of Nesfatin-1 in Synovium

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Due to limitations in the available and small sample volume, 5 synovium samples in each group were harvested, fixed in 4% paraformaldehyde for 48 h, and embedded in paraffin. Serial sections (4 μm) were cut. Immunohistochemistry for nesfatin-1 was performed using the SP-9000 Histostain-Plus kit (ZSGB Bio, China), according to the manufacturer’s instructions. Samples were visualized using a digital pathology slide scanner (3DHISTECH Ltd., The Digital Pathology Company, Budapest, Hungary). Image-ProPlus Software (Media Cybernetics, Silver Spring, MD, USA) was used to calculate the integral optical density of the sections.
Zhang S., Rong G., Xu Y, & Jing J. (2021). Elevated Nesfatin-1 Level in Synovium and Synovial Fluid is Associated with Pro-Inflammatory Cytokines in Patients with Rheumatoid Arthritis. International Journal of General Medicine, 14, 5269-5278.
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3

Immunohistochemical Analysis of NUCB2/Nesfatin-1 in Gastric Cancer

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The tissue samples were embedded in paraffin and cut into 4‐μm sections. Immunohistochemical staining of NUCB2/nesfatin‐1 in GC tissues was performed with the SP‐9000 Histostain‐Plus kits (ZSGB Bio) according to the manufacturer's instructions. Anti‐human NUCB2/nesfatin‐1 monoclonal antibody (Catalogue # MAB5949) was obtained from R&D Systems. Samples were visualized by a digital pathology slide scanner (3DHISTECH, The Digital Pathology Company, Budapest, Hungary). Image‐Pro Plus Software (Media Cybernetics) was used to calculate the integral optical density (IOD) of the sections.
Ren L., Bao D., Wang L., Xu Q., Xu Y, & Shi Z. (2022). Nucleobindin‐2/nesfatin‐1 enhances the cell proliferation, migration, invasion and epithelial‐mesenchymal transition in gastric carcinoma. Journal of Cellular and Molecular Medicine, 26(19), 4986-4994.
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4

Immunohistochemistry of LC3B and P-AKT

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IHC were performed with the SP-9000 Histostain-Plus kits (ZSGB-BIO) as previous protocols. The following antibodies were used: LC3B (1:200 dilution; Cell Signaling Technology, #2775), P-AKT308 (1:500 dilution; Cell Signaling Technology, #13038) followed by diaminobenzidine (DAB) kit detection. Positive cells from three random high-power fields were calculated for statistical analysis.
Chen L., Ni Z., Huang J., Zhang R., Zhang J., Zhang B., Kuang L., Sun X., Zhang D., Su N., Qi H., Yang J., Jin M., Luo F., Chen H., Zhou S., Du X., Ouyang J., Wang Z., Xie Y., Tan Q, & Chen L. (2021). Long term usage of dexamethasone accelerating the initiation of osteoarthritis via enhancing the extracellular matrix calcification and apoptosis of chondrocytes. International Journal of Biological Sciences, 17(15), 4140-4153.
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5

Immunohistochemical Analysis of Bone Markers

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IHC was performed with the SP-9000 Histostain-Plus kits (ZSGB-BIO) according to the manufacturer’s instructions as previously described (Wang et al., 2017 (link)). Briefly, sections were deparaffinized in xylene, rehydrated in a graded series of alcohol washes and washed twice in PBS for 5 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 10 min followed by antigen retrieval using 0.1% trypsin for 15 min. The sections were then blocked with normal goat serum for 30 min and incubated with different antibodies at 4°C overnight. On the second day, the sections were rinsed with PBS after incubation with horseradish peroxidase conjugated secondary antibodies for 30 min at 37°C. Finally, the sections were stained with a diaminobenzidine kit. Primary antibodies against the following proteins were used: ALK5 (1:100, Santa Cruz Biotechnology, Cat# sc-398, RRID: AB_632493), runt-related transcription factor 2 (RUNX2; 1:200, Santa Cruz Biotechnology, Cat# sc-390715, RRID: AB_2637033), Collagen II (Col II; 1:200, Millipore, Cat# MAB8887, RRID: AB_2260779), and matrix metalloproteinase 13 (MMP13; 1:200, Proteintech, Cat# 18165-1-AP, RRID: AB_2144858).
Wang Q., Tan Q., Xu W., Kuang L., Zhang B., Wang Z., Ni Z., Su N., Jin M., Li C., Jiang W., Huang J., Li F., Zhu Y., Chen H., Du X., Chen D., Deng C., Qi H., Xie Y, & Chen L. (2018). Postnatal deletion of Alk5 gene in meniscal cartilage accelerates age-dependent meniscal degeneration in mice. Journal of cellular physiology, 234(1), 595-605.
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6

Immunohistochemical Analysis of B7-H3 Expression

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Clinical specimens were used for immunohistochemical staining analyses. Paraffin-embedded tissues were cut into 4-μm sections and incubated with the rabbit anti-human B7-H3 polyclonal antibody (1:50 dilution) overnight at 4°C. SP-9000 Histostain-Plus kits (ZSGB-BIO, Beijing, China) were used according to the manufacturer’s instructions. The brown staining in the cytoplasm was read as positive reactivity for B7-H3. Scoring was measured according to the cell cytoplasm staining pattern: 0, no cytoplasmic staining;1, weak cytoplasmic staining; 2, moderate cytoplasmic staining; and 3, strong cytoplasmic staining.
Li D., Wang J., Zhou J., Zhan S., Huang Y., Wang F., Zhang Z., Zhu D., Zhao H., Li D., Chen G., Zhu X, & Zhao X. (2017). B7-H3 combats apoptosis induced by chemotherapy by delivering signals to pancreatic cancer cells. Oncotarget, 8(43), 74856-74868.
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7

IHC Analysis of Cartilage Degeneration Markers

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Immunohistochemistry (IHC) was performed with the SP-9000 Histostain-Plus kits (ZSGB-BIO) according to the manufacturer’s instructions. The following antibodies were used: ALK5 (1:100; Santa Cruz Biotechnology), pSmad3 (1:100; Cell Signaling Technology), MMP13 (1:200; Proteintech), ADAMTS5 (1:200; Abcam), AGGRECAN (1:200; Millipore), Cleaved caspase 3 (1:100; Boster), PRG4 (1:500; Abcam), pCREB (1:100; Abcam).
Wang Q., Tan Q.Y., Xu W., Qi H.B., Chen D., Zhou S., Ni Z.H., Kuang L., Guo J.Y., Huang J.L., Wang X.X., Wang Z.Q., Su N., Chen L., Chen B., Jiang W.L., Gao Y., Chen H.G., Du X.L., Xie Y.L, & Chen L. (2017). Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice. Osteoarthritis and cartilage, 25(11), 1868-1879.
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8

Immunohistochemical Analysis of Cartilage Markers

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For immunohistochemical staining, the experiment was performed using SP-9000 Histostain-Plus kits (Zsgb Bio, China) according to the manufacturer’s protocol. Briefly, endogenous peroxidase activity was quenched with 3% (v/v) hydrogen peroxide in methanol, and then antigen retrieval was done via 0.1% trypsin, followed by blocking process with goat serum for 60 min. Sections were incubated overnight at 4 °C with the following primary antibodies: p16INK4a (1:500; Abcam, ab54210), MMP-13 (1:200; Abcam, ab39012), type II collagen (1:300; Abcam, ab34712) and HMGB1 (1:500; Abcam, ab18256). Antibodies were diluted in 3% BSA dissolved in PBST with 2.5% Triton X-100. After rinsing with PBS for three times, sections were incubated with homologous biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin-biotin. The total number of positively stained cells on average 3 views of each section were counted using Image-Pro Plus version 5.1 software (Media Cybernetics) for histomorphometric measurements. The percentage of positive cells was calculated and the relative fold change was determined.
Yang H., Chen C., Chen H., Duan X., Li J., Zhou Y., Zeng W, & Yang L. (2020). Navitoclax (ABT263) reduces inflammation and promotes chondrogenic phenotype by clearing senescent osteoarthritic chondrocytes in osteoarthritis. Aging (Albany NY), 12(13), 12750-12770.
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9

Immunohistochemical Profiling of IVD Pathogenesis

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Immunohistochemistry was performed using SP-9000 Histostain-Plus kits (ZSGB-BIO, Beijing, China) according to a previous study.63 (link) Briefly, decalcified IVD tissues were deparaffinized with xylene, and endogenous peroxidase activity was quenched with 3% H2O2. Antigen retrieval was performed with 0.1% trypsin, and normal goat serum was used for blocking for 30 min. Sections were incubated overnight with anti-Hif1α (1:200 dilution; Abcam, MA, USA), anti-Hif2α (1:200 dilution; Abcam, MA, USA), anti-VHL (1:200 dilution; Abcam, MA, USA), type X collagen (COLX) (1:400 dilution; Abcam, MA, USA), anti-osteocalcin (1:100 dilution; Santa Cruz Biotechnology), anti-RUNX2 (1:100 dilution; Santa Cruz Biotechnology), anti-VEGF (1:200 dilution; Abcam, MA, USA), anti-GLUT1 (1:200 dilution; Millipore, Billerica, MA, USA), anti-LDHA (1:200 dilution; Abcam, MA, USA), and anti-PDK1 (1:200 dilution; Abcam, MA, USA). After rinsing with PBS, a horseradish peroxidase (HRP)-conjugated secondary antibody was applied and stained with a DAB kit.
Wang Z., Chen H., Tan Q., Huang J., Zhou S., Luo F., Zhang D., Yang J., Li C., Chen B., Sun X., Kuang L., Jiang W., Ni Z., Wang Q., Chen S., Du X., Chen D., Deng C., Yin L., Chen L, & Xie Y. (2022). Inhibition of aberrant Hif1α activation delays intervertebral disc degeneration in adult mice. Bone Research, 10, 2.
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10

Immunohistochemistry for Cartilage Markers

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Immunohistochemistry (IHC) were carried out using SP-9000 Histostain-Plus kits (Zsgb Bio) as previous protocols 13 . Incubated with antibody specific to Aggrecan (1:200 dilution; Abcam), type II collagen (Col II) (1:400 dilution; Chondrex), Adamts5 (1:200 dilution; Abcam), MMP13 (1:200 dilution; Abcam), Col X (1:200 dilution; Millipore) and FGFR3 (1:200 dilution; Santa Cruz Biotechnology) followed by diaminobenzidine (DAB) kit detection, all antibodies were solvated with antibody diluent (Origene).
Tan Q., Chen B., Wang Q., Xu W., Wang Y., Lin Z., Luo F., Huang S., Zhu Y., Su N., Jin M., Li C., Kuang L., Qi H., Ni Z., Wang Z., Luo X., Jiang W., Chen H., Chen S., Li F., Zhang B., Huang J., Zhang R., Jin K., Xu X., Deng C., Du X., Xie Y, & Chen L. (2018). A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice. Osteoarthritis and cartilage, 26(12).
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