BALF was obtained as previously described (14 (link)). ELISA kits were used to measure serum leptin (Invitrogen, Carlsbad, CA), triacylglycerol (TAG, Pointe Scientific, Canton, MI), insulin (Uppsala, Sweden) and adiponectin (Abcam, Cambridge, UK). Blood glucose was measured via a 6h or 14h fast as described in figure legends. Further, BALF albumin was measured with the Mouse Albumin ELISA Kit (Genway Biotech, Inc., San Diego, CA). Total protein in BALF was measured via standard BCA assay (BCA kit, Sigma-Aldrich, St Louis, MO). BALF cytokines (IL-4, IFN-γ, MCP-1, RANTES, KC, IL-17A, IL-10 and TNF-α) were measured using a BioRad Bio-Plex assay (Hercules, CA) per manufacturer’s instructions. IL-4 was not measureable above the lower limit of the assay.
Insulin
Manufactured by Abcam
Sourced in United States, United Kingdom
Insulin is a peptide hormone produced by the pancreas. It is a key regulator of glucose metabolism, promoting the uptake and storage of glucose in cells. Insulin plays a critical role in maintaining blood glucose levels within a normal range.
Automatically generated - may contain errors
Lab products found in correlation
Somatostatin, by Abcam (6 mentions)
Glucagon, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Somatostatin, by Agilent Technologies (3 mentions)
Glucagon, by Abcam (3 mentions)
Glucagon, by Agilent Technologies (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Anti mettl3, by Abcam (2 mentions)
Anti mettl14, by Merck Group (2 mentions)
Alkbh5, by Merck Group (2 mentions)
Axio imager a2 upright fluorescence microscope, by Zeiss (2 mentions)
Alexa 594, by Jackson ImmunoResearch (2 mentions)
Alexa 488, by Jackson ImmunoResearch (2 mentions)
Glucagon, by Cell Signaling Technology (2 mentions)
Proliferating cell nuclear antigen (pcna), by Thermo Fisher Scientific (2 mentions)
Ms 1499 p1, by Abcam (2 mentions)
Cy3 or dylight488 conjugated streptavidin, by Jackson ImmunoResearch (2 mentions)
Fitc conjugated affinipure donkey anti goat igg, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
38 protocols using insulin
1
Bronchoalveolar Lavage Fluid Analysis
2
Immunostaining of Mouse Pancreatic Tissue
3
Immunofluorescence of Pancreatic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence was performed as previously described [20 (link)]. Pancreatic tissue was fixed in 10% neutral buffered formalin and antigen retrieval was completed with Antigen Unmasking Solution (Vector Laboratories). Primary antibodies included ARC (Cayman), Ki67 (Abcam), and insulin (Abcam), and secondary antibodies were Alexa Fluor 488 or 568 (Invitrogen). Samples were counterstained and coverslipped with VECTASHIELD HardSet Mounting Medium with DAPI (Vector Laboratories).
4
Bovine Mammary Epithelial Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experimental samples in this research were bMECs that had been stored in our laboratory. The bMECs were purified and cultured from a healthy dairy cow according to previous work performed in our laboratory by tissue nubble culture method (10 (link)). The cells were cultured in a basal medium containing Dulbecco's modified Eagle's medium and Nutrient Mixture F-12 (DMEM/F12, HyClone, Logan, USA) containing 10% FBS (fetal bovine serum, Tian hang, Zhejiang, China), 25 μL hydrocortisone (25 g/ml, Abcam, Cambridge, UK), and 25 μL insulin (0.25 EU/mg, Abcam, Cambridge (HyClone, Logan, USA).
5
Immunostaining of Mouse Pancreatic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse pancreas was collected and fixed in 4% formaldehyde at 4°C overnight, followed by paraffin embedding. Five-micron thick slides were cut and subjected to immunostaining. Slides were heated in 10mM sodium citrate, followed by blocking with donkey serum and incubated with various primary antibodies: Ki67 (#550609, BD, USA), Anti-METTL3 (#195352, Abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), ALKBH5 (#HPA007196, Sigma, USA), PDX1 (#5679, Cell Signaling, USA), Insulin (#ab7842, abcam, USA), Glucagon (#G2654, Sigma, USA), Somatostatin (#ab64053, abcam, USA). Specific signal was detected by using fluorescence-conjugated secondary antibodies (Jackson Immunoresearch, Alexa 488, Alexa 594, and AMCA) (please see Reporting Summary for details on antibodies used). Images were captured using Zeiss Axio Imager A2 upright fluorescence microscope. The β-cell mass was calculated by generating the ratio of the cross-sectional area of total number of pixels of Insulin positive cells to the cross-sectional area of total number of pixels of the pancreatic tissue, multiplied by the pancreas weight of the mouse. We evaluated β-cell proliferation by co-immunostaining one section of each pancreas sample with Ki67, Insulin and DAPI and counting 1000–2000 cells in a blinded manner by a single observer33 (link),34 (link),37 (link).
6
Histological and Immunohistochemical Analysis of Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were fixed with 4% paraformaldehyde overnight, paraffin-embedded and sectioned (4–5 μM) as previously described9 (link). Sections were deparaffinized and stained with H&E for initial histology analysis. For further immunohistochemistry analysis, we used antibodies to the following proteins: PCNA (1:300; Invitrogen,133940), APC (1:100; Abcam), E-cadherin (1:300; BD Biosciences Pharmingen, 610182), GFP (1:100; Abcam, ab290), p53 (1:100; Santa Cruz, sc-1311), Kras (1: 100; Abcam, ab16907), Villin (1:100; NeoMarkers, MS-1499-P1), SMA (1:200; Abcam, ab5694), Pdx1 (1:100; Abcam, ab47267), Amylase (1:100; Santa Cruz, sc-31869), Somatostatin (1:200; Abcam, ab103790), Insulin (1:100; Abcam, ab63820), Glucagon (1:100; Cell Signaling, #2760), PanCK (1:200; Sigma, C2931), CK19 (1:200; Abcam, ab52625). Secondary antibodies used were: Cy3- or Dylight488-conjugated Streptavidin (1:500; Jackson ImmunoResearch Laboratories), Alexa Fluor 488 goat anti-mouse (1:1,000; Invitrogen), Alexa Fluor 488 goat anti-rabbit (1:1,000; Invitrogen), FITC-conjugated Affinipure Donkey anti-goat IgG (1:500; Jackson ImmunoResearch Laboratories)
7
Quantifying Functional Factors via IHC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical (IHC) staining was performed on the sectioned tissue to assess the quantity of functional factors. After blocking in phosphate‐buffered saline containing 5% goat serum for 30 minutes at room temperature (20‐25°C), the sections were incubated overnight at 4°C with primary antibodies: insulin, CD68, and caspase‐3 (1:200; Abcam, Cambridge, UK). The following day, signal amplification was performed with horseradish peroxidase–conjugated goat anti‐rabbit IgG secondary antibody (1:500; ZhongshanJinqiao Biotechnology Co., Ltd, Beijing, China). The expression levels of factors were quantified using integrated optical density values generated by Image‐Pro Plus 6.0 software.
8
Pancreatic Tissue Analysis and Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pancreatic tissue was prepared and stained as previously described14 (link). Primary antibodies were ARC (Cayman Chemical), insulin (Abcam), BrdU (Roche), cleaved caspase-3 (Cell Signaling Technology), and α-tubulin (Sigma-Aldrich). Alexa Fluor 488 and 568 (Invitrogen) of corresponding species were used for recognition of primary antibodies. All images were collected with Axio Observer.Z1 microscope (Zeiss). Islet morphology was designated as abnormal if >25% of perimeter was jagged on hematoxylin and eosin-stained sections. All islets (~85–100) in pancreatic section were scored. β-cell area was determined by calculating the number of β-cell nuclei (insulin-positive cells) in an islet divided by the respective islet area (quantified with ImageJ; National Institutes of Health). A minimum of five islets were scored per mouse. For BrdU staining, BrdU 2.2 mg/ml was included in the drinking water for 4 days prior to sacrifice, and tissue sections were treated with 2 M HCl for 10 minutes at room temperature immediately following antigen retrieval. TUNEL staining was performed as previously described14 (link). Slides were coverslipped with VECTASHIELD mounting media with DAPI (Vector Laboratories).
9
Histological and Immunohistochemical Analysis of Organoids
10
Immunohistochemical Analysis of Islet Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin wax; 5-µm-thick sections were prepared for immunostaining. Immunohistochemistry and dual immunofluorescence labeling were performed on tissue sections as described previously (14, 16, 20 (link)
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!