Serial 4 μm thick paraffin sections were used for immunohistochemistry with antibodies specific for IGF2BP3 (ab179807, Abcam, USA, 1:100), E2F3 (DF12390, Affinity Biosciences, Changzhou, China, 1:50), and Ki67 (AF1738, Beyotime, Shanghai, China, 1:50). A streptavidin peroxidase (SP) immunohistochemical kit (Maixin, Fuzhou, China) was used according to the instructions. Images were taken with a Nikon microscope (Eclipse Ci, Nikon Ltd, Japan). Image-Pro Plus 6.0 was employed to analyse the levels of protein expression by calculating the values of mean integrated optical density (integrated optical density/area) for statistical analysis [47 (link)].
Sp immunohistochemical kit
Manufactured by Maixin Group
Sourced in China
The SP immunohistochemical kit is a laboratory tool designed for the detection and visualization of specific proteins or antigens in biological samples. It utilizes the streptavidin-peroxidase (SP) method to amplify the signal and enhance the sensitivity of the immunohistochemical staining process. The kit includes all the necessary components to perform the staining procedure, such as primary antibodies, biotinylated secondary antibodies, and the streptavidin-peroxidase conjugate.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e600 microscopy system, by Nikon (2 mentions)
Ab179807, by Abcam (1 mentions)
Af1738, by Beyotime (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Dab detection kit, by Vector Laboratories (1 mentions)
Anti rankl antibody, by Abcam (1 mentions)
Proliferating cell nuclear antigen (pcna), by Maixin Group (1 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
Cetuximab, by Merck Group (1 mentions)
Dab kit, by Maixin Group (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
6 protocols using sp immunohistochemical kit
1
Immunohistochemical Analysis of Protein Expression
2
Histological Examination of Kidney Tissue
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Hematoxylin–eosin, periodic acid–Schiff (PAS), and Masson’s trichrome stainings were performed by conventional histochemical methods described previously [25 (link)]. After the mice were euthanized, full-thickness biopsies were taken and fixed in 4% formaldehyde, and then embedded in paraffin. Tissue slices were subjected to hematoxylin–eosin staining. For PAS staining, the kidneys were harvested and fixed in 10% formalin; 5-μm thick sections were stained with PAS reagent. Immunohistochemistry was performed in paraffin sections and detected in cortical sections with a specific primary antibody and the ABC staining kit, and visualized with the DAB detection kit (both kits from Vector Laboratories Inc., Burlingame, CA, USA). The primary antibody used in the present study was RAS (Sigma; Maixin, Fuzhou, China). After being incubated with the secondary antibody (Proteintech Group, Chicago, IL, USA), 2-μm thick sections were developed with an SP immunohistochemical kit (Maixin, China) to produce a brown product and counterstained with hematoxylin. Histologic evaluation was performed using a Nikon Eclipse E600 microscopy system (Nikon Instruments Inc., Melville, NY, USA) without knowledge of the identity of the various groups. The slides were coded and examined by a pathologist blinded to the study protocol to identify histological alterations.
3
Immunohistochemical Analysis of RANK and RANKL in Gastric Carcinoma
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Formalin-fixed, paraffin-embedded primary gastric carcinoma tissues were cut into 3-mm sections. The IHC method is discussed in our previous study (20 (link)). S-P immunohistochemical kit and 30-diamino-benzidine tetrahydrochloride (DAB) kit were obtained from Maixin Bio (Fuzhou Maixin Biological Technology Ltd., China). Immunohistochemical staining was performed using the following antibodies: anti-RANK antibody from RD Company and anti-RANKL antibody from Abcam (USA). Sections were observed through microscopy (×20 and ×40) by two independent pathologists. From each section, 5 visual fields were randomly selected and scoring was done according to the percentage of positive cells and the staining intensity. Positive cells of <10, 10-25, 26-50, 51-75, >76% were recorded as 0, 1, 2, 3, and 4, respectively. A score >2 was considered as high expression, 0-2 as low expression.
4
Kidney Tissue Morphology Analysis
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The kidneys were harvested and fixed in 10% formalin. 5 μm thick sections were stained with periodic acid-Schiff (PAS) reagent. Immunohistochemistry was performed in paraffin sections using a high-temperature-heating antigen retrieval method. Primary antibody used in the present study was proliferating cell nuclear antigen (PCNA, Maixin, Fuzhou, China). After being incubated with the secondary antibody (Proteintech Group, Chicago, IL, USA), 2 μm thick sections were developed with SP immunohistochemical kit (Maixin, Fuzhou, China) to produce a brown product and counterstained with hematoxylin. Histologic evaluation was performed using a Nikon Eclipse E600 microscopy system without knowledge of the identity of the various groups.
5
Cetuximab and RANKL Signaling Modulation
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Cetuximab was purchased from EMD Millipore (Billerica, MA, USA). Recombinant sRANKL and rOPG was purchased from CytoLab/PeproTech Asia (USA). PP2 was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Dasatinib was obtained from Selleck Chemicals (Houston, TX, USA). S-P immunohistochemical kit and 3,3′-diaminobenzidine tetrahydrochloride kit were obtained from Maixin Bio (Fuzhou Maixin Biological Technology Ltd., Fujian, People’s Republic of China).
6
Immunohistochemical Staining of CD11c
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Paraffin-embedded specimens were deparaffinized with xylene, rehydrated using graded ethanol, then boiled in sodium citrate-hydrochloric acid buffer in a pressure cooker for 10 minutes, and cooled for 30 minutes at room temperature to expose antigenic epitopes. Then, the samples were blocked with 5% normal goat serum in 1% bovine serum albumin in PBS for 30 minutes at room temperature. The slides were then incubated with polyclonal rabbit anti human-CD11c antibody at a dilution of 1:500 and incubated overnight at room temperature. After being washed thrice with 0.05% Tween 20 in PBS for 10 minutes, slides were stained with secondary antibody for 1 hour and then washed thrice with 0.05% Tween in PBS for 10 minutes again. Slides were then stained with hematoxylin and washed with running water. For immunohistochemical staining, the samples were then processed using the SP immunohistochemical kit (Maixin, Fuzhou, China), and the immunoreactive proteins were detected using a DAB kit (Maixin, Fuzhou, China). After all the stains, slides were dehydrated with ethanol followed by dimethylbenzene. Finally, after being mounted with cover slides, samples were observed under the microscope (Nikon, Eclipse 80i). The number of positive cells per field of each index was obtained by counting five separated fields (×100) with Image-Pro Plus software (v. 5.0).
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