The specimens were fixed with 10% formalin, embedded in paraffin, and sliced to obtain 5-µm serial sections. Ultra-sensitive TM SP (mouse/rabbit) IHC kit (Maixin, China, Cat# KIT-9720) was used to detect the expression of TRIB3. A breast cancer paraffin section was used as the TRIB3 positive control, and phosphate buffered saline (PBS) was used as the negative control. Rabbit TRIB3 polyclonal antibody (Proteintech, Wuhan, China, Cat# 13300-1-AP) was used at a dilution of 1:1000. The presence of buffy granules in the cell membrane and cytoplasm was defined as positive. The dyeing intensity was recorded as 0 (no coloration), 1 (yellow), 2 (brownish-yellow), and 3 (brown). The percentage of stained cells throughout the section was scored as follows: 0 for < 5%, 1 for 5 to 25%, 2 for 26 to 50%, 3 for 51 to 75%, and 4 for > 75%. The final score was obtained by multiplying the intensity score and the percentage score. A final score of 0–2 indicated negative expression (−), 3–4 indicated weak positive expression (+), 5–8 indicated positive expression (++), and 9–12 indicated strong positive expression (+++). Each section was independently evaluated by two senior pathologists. If the specimen evaluation did not show consistency between the two pathologists, the sample was then evaluated by a third pathologist.
Ultrasensitive sp mouse rabbit ihc kit
Manufactured by Maixin Group
Sourced in China
The UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit is a comprehensive immunohistochemistry (IHC) staining kit designed for the detection of mouse and rabbit primary antibodies in formalin-fixed, paraffin-embedded (FFPE) tissue sections. The kit provides all the necessary reagents for antigen retrieval, blocking, primary antibody incubation, and detection, enabling reliable and sensitive visualization of target proteins.
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Lab products found in correlation
Diaminobenzidine substrate kit, by Maixin Group (1 mentions)
Balb c nude mice, by Beijing Huafukang Biotechnology (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Ab194201, by Abcam (1 mentions)
Ab176005, by Abcam (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Picrosirius red, by Solarbio (1 mentions)
Colorimetric tunel apoptosis assay kit, by Beyotime (1 mentions)
Masson s trichrome stain kit, by Solarbio (1 mentions)
Alanine aminotransferase assay kit, by Nanjing Jiancheng (1 mentions)
Aspartate aminotransferase assay kit, by Nanjing Jiancheng (1 mentions)
Ab97910, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Ab200587, by Abcam (1 mentions)
17 protocols using ultrasensitive sp mouse rabbit ihc kit
1
Immunohistochemical Analysis of TRIB3 Expression
2
Immunohistochemical Analysis of Tumor Tissues
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For immunohistochemical analysis, tumor tissues were fixed using 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned at 4 μm, and mounted on glass slides. The slides were deparaffinized, rehydrated, and incubated with 3% H2O2. A primary antibody was added, and the slides were incubated at 4 ℃ overnight. Subsequently, a secondary IgG antibody with an HRP label was added. Staining was performed using a 3, 3-diaminobenzidine (DAB) substrate kit for peroxidase reaction and counterstained with hematoxylin. The UltrasensitiveTM SP (Mouse/Rabbit) IHC kit (Mai-xin Biotechnology Co., Fuzhou, China) was applied to IHC detection, strictly following the manufacturer’s protocol. Finally, images were observed and captured with an optical microscope.
3
Immunohistochemical Analysis of BAP18 in Prostate Tissue
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Sections of prostate tissue specimens were prepared from clinical prostatectomy specimens in the First Hospital of China Medical University. The procedure was performed as previously described (28 (link),31 (link)). Tissue sections of formalin-fixed, paraffin embedded tissue blocks were dewaxed in xylene, rehydrated in decreasing concentrations of ethanol, and blocked for endogenous peroxidase activity using 3% hydrogen peroxide. Antigen retrieval was carried out in citrate buffer (pH 6.0) at 130°C for 2 min. Then, non-specific antibody binding was blocked by incubating with goat serum for 15 min at room temperature. Slides were incubated with anti-BAP18 (1:100) overnight at 4°C. After washing, slides were incubated with biotinylated goat anti-rabbit/mouse immunoglobulin, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex (UltraSensitiveTM SP(Mouse/Rabbit)IHC Kit, Maixin Bio). The sections were developed with the diaminobenzidine substrate kit (Maixin Bio) and counterstained with hematoxylin. Images were taken with an Olympus microscope. This study was approved by the Ethics Committee of the Medical Faculty of the China Medical University.
4
Immunohistochemical Analysis of Murine Xenograft
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BALB/c nude mice (4–6 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd., China. Animals were maintained in specific pathogen-free conditions and environment under controlled conditions of light and humidity. Animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory. The mice were randomly divided into 4 groups (5 animals/group) that were designated as the SMMC-7721 group, the NC group, the MR1-D4 group and the MR1-D9 group. Cells (2 × 106) were subcutaneously injected into the right flank of each mouse. On day 21 post-injection, the tumors were dissected, fixed in 10% neutral-buffered formalin and embedded in paraffin for immunohistochemical staining using antibodies against MUC1 (GP1.4), p-JNK, p-Smad3L (Ser-213), c-Myc, and an UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (MaiXin.BIO., Fuzhou, China). Liver tissues from hemangioma patients and HCC patients were used similarly to the mouse tumors. The sections were examined on an inverted fluorescence microscope (IX71; Olympus).
5
Immunohistochemistry Protocol for Protein Expression
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All samples were fixed in 10% neutral‐buffered formalin and embedded in paraffin. Antibodies were the same as Western blotting and using an UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (MaiXin.BIO., Fuzhou, China). The sections were examined using an IX71 microscope (Olympus, Tokyo, Japan). For quantitative analyses, areas of positive staining were analyzed using Image‐Pro Plus 6.0 (Media Cybernetics, Inc. Silver Spring, MD, USA). Five fields of view for each section were randomly selected and images acquired, the Integral Optical Density Sum (IOD sum) and the corresponding area were measured. The index of density (mean) = IOD sum/area was used to evaluate the expression of the proteins.
6
Tissue Microarray for Immunohistochemical Analysis
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All tissue specimens were fixed in 4% paraformaldehyde, embedded in paraffin, then dehydrated in an alcohol gradient and xylene until they were transparent. Paraffin-embedded tissue samples were used to construct a tissue microarray using the Manual Tissue Microarrayer (Quick-ray, UniTMA, Seoul, South Korea). IHC was performed on tissue microarray samples using routine experimental procedures 17 (link). The primary antibodies were rabbit anti-RIOK1 (N-terminal) polyclonal antibody (1:100; ab176005, Abcam, Cambridge, UK) and AKT1 polyclonal antibody (1:100; ab194201, Abcam). An UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (KIT-9710, Maixin, Shenzhen, China) was used to label tissue. Scoring criteria for IHC were described in our previous publication 17 (link).
7
Immunohistochemical Quantification of KRT80
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Paraffin-embedded tissue samples from each group were fixed in 10% formalin and sectioned continuously at a thickness of 5 μm after conventional paraffin embedding. KRT80 expression was detected using an ultra-sensitive TM SP (mouse/rabbit) IHC kit (Maixin, China, Cat# KIT-9720). Rabbit KRT80 polyclonal antibody (Proteintech, Wuhan, China, Cat# 16835-1-AP) was used at a 1:500 dilution. Brown and yellow granules were observed in the cytoplasm, and were considered to be positive staining. A staining intensity score was adopted, with 0 indicating no coloring, 1 indicating light yellow, 2 indicating brown yellow, and 3 indicating brown. The percentage of stained cells in a visual field was scored according to the following: 0 (< 5%), 1 (5 ~ 25%), 2 (26 ~ 50%), 3 (51 ~ 75%), and 4 (> 75%). The final score was calculated as the product of the staining score and percentage of stained cells in a visual field, and was denoted as follows: 0 ~ 2 (-), 3 ~ 4 (+), 5 ~ 8 (+ +), and 9 ~ 12 (+ + +). Based on these scores, 0 ~ 4 was designated as the 'low expression group' and 5 ~ 12 as the 'high expression group'. Each sample was scored by two independent observers, and disagreements were resolved by a third independent observer.
8
Immunohistochemical Evaluation of PD-L1, CD8, and PD-1
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Tissue samples were prepared and preserved in FFPE. Serial 4-µm-thick sections were prepared through deparaffination in xylene and dehydration with graded alcohols. For IHC staining, the procedure was performed followed by the standard procedure for the UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (#9710, Maixin Biotech, Fuzhou, Fujian, China). Firstly, heat-induced antigen retrieval in citrate buffer was conducted. After blocking with bovine serum albumin (BSA), the sections were incubated with the following primary antibodies at 4 °C overnight: PD-L1 (#13684, clone E1L3N, CST), CD8 (MAB-0021, clone c8/144B, Maixin Biotech, Fuzhou, Fujian, China), PD-1 (#86163, clone D4W2J, CST). Next, the sections were treated with second antibody-peroxidase-linked polymers and visualized with 3,3-diamino-benzidine tetrahydrochloride (DAB; Maixin Biotech, China).
9
Immunohistochemical Analysis of Apoptotic Markers
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UltrasensitiveTM SP (Mouse/Rabbit) IHC Kit (Maixin Biotechnology Co., Fuzhou, China) was used to perform immunohistochemistry. The tumor tissue was made into 5 µm frozen sections. After adding peroxidase blocker and non-specific staining blocker sequentially, the sections were incubated with the primary antibodies (Caspase-3: 1:1000, Cell Signaling Technology; Bcl-2: 1:800, Cell Signaling Technology) at 4°C overnight and then incubated with biotin-labeled goat anti-mouse/rabbit IgG at room temperature for 10 minutes. Streptomyces anti-biotin proteinperoxidase was added into the sections and incubated for 10 minutes. The neutral resin was used to seal the sections after DAB and hematoxylin staining and the microscope was used to observe.
10
Immunohistochemical Staining Protocol
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The UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (Maixin Biotech, Fuzhou, China) which uses streptavidin-biotin amplification method was applied to perform immunohistochemical study. After deparaffinizing the 4μm tissue samples, we blocked endogenous peroxidase activity with 0.3% hydrogen and incubated sections with 0.03 mol/l citrate buffer (pH 6.0) in a pressure cooker under 121°C. Nonspecific reaction was blocked by using 10% bovine serum (Biosharp, Hefei, China) for 30 min. Finally, sections were incubated with primary antibody overnight at 4°C and in turn detected with 3.3′-diaminobenzidine (DAB) solution. The primary antibodies we used were listed in Table 1.
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