Livers containing islet grafts were procured from recipient mice, fixed in 10% formalin neutral buffer solution, embedded in paraffin, and cut into 5 μm-thick sections. Hematoxylin and eosin (H&E) staining was preformed according to standard protocols. Immunohistochemical staining was performed as previously reported.25 (link) Sections were incubated overnight at 4°C with the following primary antibodies: antiinsulin (1:2000; Proteintech, Tokyo, Japan), anti-CD4 (1:100; D7D2Z, Cell Signaling Technology), anti-CD8α (1:400; D4W2Z, Cell Signaling Technology), anti-CD31 (1:100; Cell Signaling Technology), and anti-F4/80 (1:50; Novus Biologicals), followed by incubation for 40 min with a biotinylated secondary antibody diluted 1:300 in PBS. Sections were washed with PBS before addition of avidin-biotin-peroxidase complex (1:100 in biotinylated secondary antibody; ABC-Elite, Vector Laboratories, Burlingame, CA) for 50 min. After washing in PBS, the color reaction was carried out using diaminobenzidine and nuclei were counterstained with hematoxylin. The number of positively stained lymphocytes that had infiltrated into the islet grafts was counted in all lobes of the liver (at the maximum cross section, which contained about 10 islets in total). The areas of liver sections that stained positively for insulin were measured and analyzed using NIH Image J software.
Anti insulin
Manufactured by Proteintech
Sourced in Japan
Anti-insulin is a lab equipment product from Proteintech. It is an antibody that binds to and neutralizes the insulin hormone. The core function of Anti-insulin is to facilitate the study and analysis of insulin and its role in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti glucagon, by Proteintech (2 mentions)
D7d2z, by Cell Signaling Technology (1 mentions)
D4w2z, by Cell Signaling Technology (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Anti f4 80, by Novus Biologicals (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Dylight 488 anti rabbit igg, by Vector Laboratories (1 mentions)
Dylight 594 anti mouse, by Vector Laboratories (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
3 protocols using anti insulin
1
Histological Analysis of Islet Grafts in Liver
2
Insulin and Glucagon Expression in Pancreatic Tissue
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Pancreatic sections were incubated with 5% BSA for 1h at room temperature and then incubated overnight with the anti-insulin (Proteintech, 15848-1-AP), and anti-Glucagon (Proteintech, 15954-1-AP) at 4°C. The next day, sections were stained with the secondary antibody for 1 hour at room temperature in the dark. The nucleus was stained with 4, 6 diamidino-2-phenylindole (DAPI) at room temperature for 5 minutes. The tissue sections and cells were imaged under a fluorescence microscope (BX61, Olympus, Japan).
3
Quantifying Pancreatic Islet Morphology
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All samples were imaged using an Olympus BX41 microscope. Image processing, quantification of pancreatic islet areas, and oil red staining were performed using ImageJ (39 ).
For immunofluorescence, deparaffinized and rehydrated sections were subjected to a citrate-based antigen retrieval. After permeabilization and blocking, sections were incubated with primary antibodies (anti-insulin (Cat #66198-1-Ig, Proteintech Group, Inc); anti-glucagon (Cat # 15954-1-AP,Proteintech Group, Inc)) overnight at 4 °C and incubated with appropriate fluorescent secondary antibodies (DyLight 488 Anti-Rabbit IgG and DyLight 594 Anti-Mouse, Vector Laboratories) for detection. Cell nuclei were stained with DAPI.
In the sections stained for insulin and glucagon, the borders of each pancreatic islet were identified morphologically and marked using the pen tool in ImageJ. The area of each islet was calculated with ImageJ software using the set scale function corresponding to the 20x scale bar. Insulin-positive nuclei were counted based on fluorescent signal. All islets with an area >566 μmˆ2 (corresponding to a diameter of >27 μm), approximately corresponding to islets containing a minimum of eight nuclei, were regarded for analysis.
For immunofluorescence, deparaffinized and rehydrated sections were subjected to a citrate-based antigen retrieval. After permeabilization and blocking, sections were incubated with primary antibodies (anti-insulin (Cat #66198-1-Ig, Proteintech Group, Inc); anti-glucagon (Cat # 15954-1-AP,Proteintech Group, Inc)) overnight at 4 °C and incubated with appropriate fluorescent secondary antibodies (DyLight 488 Anti-Rabbit IgG and DyLight 594 Anti-Mouse, Vector Laboratories) for detection. Cell nuclei were stained with DAPI.
In the sections stained for insulin and glucagon, the borders of each pancreatic islet were identified morphologically and marked using the pen tool in ImageJ. The area of each islet was calculated with ImageJ software using the set scale function corresponding to the 20x scale bar. Insulin-positive nuclei were counted based on fluorescent signal. All islets with an area >566 μmˆ2 (corresponding to a diameter of >27 μm), approximately corresponding to islets containing a minimum of eight nuclei, were regarded for analysis.
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