Paraffin sections were incubated overnight with primary antibodies in 1% horse serum albumin (PK-6200, Vector) at 4°C. The next day, the samples were washed with PBS and incubated with secondary antibodies at 25°C for 1 h. After washing with PBS, the sections were developed using ImmPACT® NovaRED® Substrate, Peroxidase (HRP) (SK-4805, Vector) for color reaction, and then observed under a microscope. The number of positive cells in 30 complete crypts was counted and expressed as the mean ± SD. Three mice were used in each group. The antibodies used were anti-BrdU (5292, CST), anti-Ki67 (9129, CST), anti-Cyclin D1 (2978, CST), anti-Lgr5/GPR49 (MAB8240, R&D Systems), anti-Sox9 (82630, CST), anti-Lysozyme (ab108508, Abcam), anti-Chromogranin A (GTX113165, GeneTex), and anti-Muc2 (GTX100664, GeneTex).
Mab8240
Manufactured by R&D Systems
MAB8240 is a monoclonal antibody product developed and manufactured by R&D Systems. It is designed for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab108508, by Abcam (1 mentions)
Pk 6200, by Vector Laboratories (1 mentions)
Gtx100664, by GeneTex (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa flour 647 anti rat, by Thermo Fisher Scientific (1 mentions)
Fv1000 laser scanning confocal cars microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
3 protocols using mab8240
1
Immunohistochemical Profiling of Intestinal Stem Cells
2
Immunohistochemistry Analysis of Intestinal LGR5 and IL-38
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse intestinal samples were collected and fixed in PBS with 4% paraformaldehyde (Sigma-Aldrich) in PBS. After dehydration, samples were embedded in paraffin molds for sectioning. Sections (5 µm) were obtained and transferred to glass microscope slides. Slides were deparaffinized with xylene and ethanol and permeabilized with 0.1%Tween in TBS solution. After antigen retrieval with citrate buffer, slides were blocked with normal donkey serum and incubated with primary antibodies against LGR5 (clone 803420, Catalog MAB8240, R&D Systems) and IL-38 (catalog ab180898, Abcam) dilution overnight at 4 °C. Alexa flour 647 anti-rat (catalog A48272, Invitrogen) and Alexa fluor 488 anti-rabbit at 1:100 (catalog 11008, Invitrogen) were used as secondary antibodies for 1 h at room temperature. Finally, slides were coverslipped in Mowiol mounting media with DAPI. Images were taken using Olympus FV1000 laser scanning confocal/CARS microscope. IL-38 antibody produces massive background probably due to possible cross-reactivity with other interleukins expressed in the intestine (i.e., IL-36a, IL-36b, IL-36g, and IL-36RA). For this reason, a reliable analysis of IL-38 staining can be performed only in comparison with an IL-38 null sample. We tried to use this IL-38 antibody for western blot, but, at least as regards intestinal samples, we could not obtain reliable results.
3
Quantifying Lgr5 Expression in Intestine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A small section of the intestine was embedded and made into paraffin sections. Then the RNA scope in situ hybridization 2.5 HD red detection kit (Advanced Cell Diagnostic, Newark, CA, United States) was used according to the manufacturer’s instructions to process the intestinal sections. In brief, the intestinal tissue underwent target retrieval, permeabilization, hybridization of Lgr5 (MAB8240, R&D Systems), amplification, and visualization using DAB-A and DAB-B. And then the intestinal sections were observed under a microscope. The expression of Lgr5 was quantitatively analyzed according to the five-grade scoring system recommended by the manufacturer (0, no staining; 1, 1–3 dots/cell; 2, 4–10 dots/cell; 3, >10 dots/cell; 4, >15 dots/cell with >10% of dots in clusters). The H-score was calculated as: (% of grade 1 cells × 1) + (% of grade 2 cells × 2) + (% of grade 3 cells × 3) + (% of grade 4 cells × 4). In addition, a cell with one or more dots was regarded as Lgr5-positive. Three mice were used in each group.
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!