SW620 xenograft tumors were instilled with 4% formalin and immersed in the same solution before tissue processing into paraffin-embedded blocks; 4 µm sections were then cut for immunohistochemical staining. After antigen retrieval for 3 mins in citrate under high pressure, tumor tissue sections were incubated with primary antibody against MDM4 (cat. no. 04-1555, 1:300; Millipore) for immunohistochemical staining following the instructions of IHC kit (SP9001; ZsBio, Beijing, China) and DAB kit (Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China). The stained tumors were visualized by a light microscope (DTX500; Nikon Corporation, Tokyo, Japan) The MDM4 positive cells were scored by counting the number of tumor cells expressing the proteins as determined by MDM4 staining in 5 random selected fields.
Ihc kit
Manufactured by ZSGB-BIO
Sourced in China
The IHC kit is a laboratory equipment used for Immunohistochemistry (IHC) analysis. It provides the necessary reagents and protocols to detect and visualize specific proteins or antigens in tissue samples. The kit includes various components required for the IHC process, such as buffers, antibodies, and detection systems.
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Lab products found in correlation
Dab kit, by ZSGB-BIO (2 mentions)
Dtx500, by Nikon (1 mentions)
P egfr, by Santa Cruz Biotechnology (1 mentions)
Cd163, by Santa Cruz Biotechnology (1 mentions)
F4 80, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Nanozoomer s60, by Hamamatsu Photonics (1 mentions)
Ab134128, by Abcam (1 mentions)
Bx50 light microscope, by Olympus (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Pha065643, by Atlas Antibodies (1 mentions)
20 protocols using ihc kit
1
Quantification of MDM4 Expression in SW620 Xenograft Tumors
2
Immunohistochemical Staining Protocol for Paraffin-Embedded Samples
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Paraffin-embedded samples were cut into 4-μm slices, heated to 60 °C for 1 hour and dehydrated by rinsing in xylene and various concentrations of ethanol. IHC staining was conducted using an IHC kit (ZSGB-BIO, China) following the manufacturer's instructions. Briefly, antigen retrieval was performed with sodium citrate buffer, and samples were incubated in primary antibodies overnight at high humidity. Then, the slices were rinsed in PBS, and 3% H2O2 was added to reduce endogenous peroxidase activity for 20 min. After washing with PBS, the response enhancer provided by the kit was added, and the sample was incubated for 20 min. The sample was incubated in secondary antibody for 20 min at 37 °C. The slices were rinsed and incubated with diaminobenzidine working solution (20 x storage solution) for 3-6 min. Then, the slices were washed with ddH2O for 10 min, and the nuclei were stained with hematoxylin for 5 min. The slices were dehydrated and sealed with resinene.
3
Immunohistochemistry of Xenograft Tissues
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Xenograft tumor tissues were fixed, dehydrated, embedded, and sectioned according to standard procedures. Antigens were retrieved by boiling in the 10 mM citrate buffer for 3 min and then incubated in 3% hydrogen peroxide for 20 min to block endogenous peroxidase. All sections were incubated with primary antibodies at 4 °C overnight. The IHC kit (ZSGB-BIO, Beijing, China) was then used to detect the specifically bound primary antibodies.
4
Immunohistochemical Profiling of Breast Tumors
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IHC staining assay was performed as previously described37 (link),58 (link). All the patient species were obtained from Qiqihar Medical University. In addition, 33 slides (17 ERα negative and 16 ERα positive) were incubated with CHES1 and ERα antibodies; the expression levels of CHES1 and ERα were quantified according to their H-scores. The IHC Kit was purchased from ZSGB-BIO (Beijing, China). All individuals who donated the tissues for this study gave their consent in written form. The expression levels of CCND1, c-MYC, and Ki67 in tumors were staining with antibodies as indicated.
5
Multimarker Immunohistochemistry Analysis
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Tissues were fixed in 4% paraformaldehyde, sent for sectioning, and assessed with an IHC kit (ZSGB-BIO, Beijing, China). Slides were incubated at 4°C overnight (16−20 h) with antibodies reactive with Ki67 (#ab16667; Abcam), F4/80 (#ab100790; Abcam), CD163 (#sc58965; Santa Cruz), VEGF (#ab32152; Abcam), VEGFR (#ab32152; Abcam), CD34 (#ab81289; Abcam), EGFR (#sc373746; Santa Cruz), and p-EGFR (#sc-377547; Santa Cruz). Diaminobenzidine (K176810E; ZSGB-BIO, China) was used for target protein staining and H&E for nuclear staining. Images were photographed at 400× with a microscope.
6
Immunohistochemical Analysis of CD3 and CD8
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Tissue with 4% formalin fixation and their chips with paraffin embedding were cut at 5 μm, followed by immunohistochemistry (IHC) staining according to directions of IHC kit (SP9000, ZSGB-BIO) and were incubated with CD3 (99940S, CST) or CD8 (98941S, CST) primary antibodies and biotinylated secondary antibodies, respectively. The signal was detected using HRP conjugated streptavidin with the chromogenic substrate DAB. The results were imaged by Leica DM4B Imaging system and interpreted by a senior pathologist. The number of CD3 and CD8 positive cells was counted by Image-Pro Plus 6.0 software.
7
Colon Adenocarcinoma Tissue Sampling and IHC
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Total 21 fresh COAD and corresponding paracancerous colon tissue samples were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University for mRNA detection, and 91 formalin-fixed, paraffin-embedded COAD tissue samples were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University between 2012 and 2013 for IHC. The patients were enrolled based on the following inclusion criteria: (1) no radiotherapy or chemotherapy before surgery and (2) no other history of surgery. Our protocol was in accordance with the ethical guidelines of the Declaration of Helsinki and was approved by Ethical Review Committee of the First Affiliated Hospital of Chongqing Medical University. All patients signed written informed consent. IHC was conducted using IHC kit (ZSGB-BIO, China) according to the manufacturer’s protocols, and the results were evaluated based on staining intensity (0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) and extent (1, <25%; 2, 25–50%; 3, 50–75%; and 4, >75%).
8
Immunohistochemical Analysis of YAP and USP36
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Tissues were incubated with 4% paraformaldehyde, embedded in paraffin, and then cut into slices using a Semi Automated Paraffin Microtome S700 (RWD, USA). IHC staining was performed with an IHC kit (Zsbio, Beijing, China) according to the manufacturer’s protocol. We used primary antibodies against YAP and USP36. After incubations with the primary and secondary antibodies, the immunocomplex was revealed with DAB, and the nuclei were counterstained with hematoxylin. We then used a NanoZoomer Digital Pathology scanner (NanoZoomer S60, HAMAMATSU, Japan) to obtain pictures. The result was evaluated by the mean of Integrated optical density (IOD) via ImageJ Software. The primary antibodies used in the IHC analysis are also shown in Supplementary Table 3 .
9
Immunohistochemical Analysis of Ki67 and Cleaved Caspase 3
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Antibodies against Ki67 and cleaved caspase 3 were purchased from Abclonal, China and CST, America, respectively. The IHC kit was provided by ZSGB-BIO, China, and used according to the manufacturer's guidelines. Briefly, the sections were heated to deparaffinization and incubated with 3% H2O2 for the proper time. Epitope retrieval was performed by heating in sodium citrate buffer at 96 °C for 30 min. For antigen-antibody reaction, samples were incubated with rabbit anti-human Ki67 (1:100 dilution) and cleaved caspase 3 (1:1000 dilution) primary antibodies for 2 hours. The cells were washed with PBS and incubated with secondary antibody followed by DAB staining and hematoxylin counterstaining. The sections were dehydrated, soaked in xylene, mounted with neutral balsam and further fastened by enamel.
10
Immunohistochemical Analysis of Hedgehog Pathway
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Immunohistochemistry (IHC) was performed using an IHC kit (Zsbio, Beijing, China). Primary rabbit anti-human CD34 (1:100; Maxim, Fuzhou, China), rabbit anti-human Shh (1:100; Novus Biologicals), rabbit anti-human/mouse PTCH (1:100; Novus Biologicals) and rabbit anti-human Gli1 (1:100; Novus Biologicals) were used. Secondary antibodies included HRP-conjugated goat anti-rat IgG and goat anti-rabbit IgG. The specimens were independently scored by two pathologists who were blinded to the clinicopathological data. An area ratio (AR) was derived from the division of the IHC-positive area by the blank area under microscopic fields, and was applied for calculation of average levels of expression: AR = 0, 0–10% positive area; AR = 1, 10–50% positive area; AR = 2, 50–80% positive area; and AR = 3, > 80% positive area. The relative expression (RE) level was the immunostaining intensity of each sample: RE = 0, negative; RE = 1, yellow staining; RE = 2, brown staining; and RE = 3, dark brown staining. The final scores were the sum of AR and RE: scores of 0–2 were negative, 3–4 were moderate and 5–6 were high 23 (link).
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