The largest database of trusted experimental protocols

4 protocols using olfm4

1

qRT-PCR Analysis of Stem Cell Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
cDNA was prepared using a Superscript III cDNA-synthesis kit (18080-044, Thermo Scientific). RT–qPCR was performed using TaqMan probes (Life Technologies) and SYBR Green (Thermo Scientific). The mRNA expression of each gene was normalized to the expression of the housekeeping genes Tbp or Hprt1. Relative expression of gene transcripts was analysed using the 2–ΔΔCt method. The RT–PCR data were collected using QuantStudio 12K Flex Software v.1.6 (Applied Biosystems). The following TaqMan probes were used: Olfm4 (Mm01320260_m1, Thermo Scientific), Lgr5 (Mm00438890_m1, Thermo Scientific), Ascl2 (Mm01268891_g1, Thermo Scientific), Tbp (Mm00446973_m1, Thermo Scientific), Prominin-1 (Mm00477115_m1, Thermo Scientific) and Lrig-5 (Mm00456116_m1, Thermo Scientific). Primer sequences for SYBR Green are described in Supplementary Table 5.
+ Open protocol
+ Expand
2

Gastric Biopsy RNA Isolation and qRT-PCR

Check if the same lab product or an alternative is used in the 5 most similar protocols
RNA used for qRT-PCR and RNAseq was isolated from a 4-mm biopsy specimen of the gastric corpus lesser curvature. RNA was extracted in TRIzol (Thermo Fisher Scientific) and precipitated from the aqueous phase using 1.5 volumes of 100% ethanol. The mixture was transferred to a RNeasy column (Qiagen, Hilden, Germany), and the remaining steps were followed according to the RNeasy kit manufacturer’s recommendations. RNA was treated with RNase-free DNase I (Qiagen) as part of the isolation procedure. Reverse-transcription followed by qPCR was performed in the same reaction using the Universal Probes One-Step PCR kit (Bio-Rad Laboratories, Hercules, CA) and the TaqMan primers (Thermo Fisher Scientific) Cftr (Mm00445197_m1), Olfm4 (Mm01320260_m1), Wfdc2 (Mm00509434_m1), Zfp36 (Mm00457144_m1), Il33 (Mm00505403_m1), and Il13 (Mm00434204_m1) (Thermo Fisher Scientific) on a Quantstudio 6 (Thermo Fisher Scientific). mRNA levels were normalized to the reference gene Ppib (Mm00478295_m1).
+ Open protocol
+ Expand
3

Histological Analysis of Intestinal Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols
Intestinal tissues of mice and minipigs were fixed with 10% neutral buffered formalin solution, embedded in paraffin wax, and sectioned transversely at a thickness of 4 µm. The slides were stained with H&E and Congo red. Histological scores were quantified in the H&E-stained slides and assessed by the degree of the epithelial maintenance, crypt damage, vascular dysfunction, and inflammation with infiltration of inflammatory cells. To investigate radiation-induced goblet cell damage, the slides were stained with PAS. For immunohistochemical analysis, the slides were subjected to antigen retrieval for 20 min and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block endogenous peroxidase activity. After washing with PBS, the slides were blocked with 10% normal goat serum (Vector ABC Elite kit; Vector Laboratories, Burlingame, CA, USA) and incubated with primary antibodies, such as anti-Cldn3 (Invitrogen, Carlsbad, CA, USA), villin (Abcam, Cambridge, UK), Mpo (Abcam), ki-67 (Acris), Olfm4 (Invitrogen), and CD68 (Abcam) antibodies. Additionally, the slides were incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA, USA) for 60 min. The peroxidase reaction was developed using diaminobenzidine substrate (Dako) prepared according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin.
+ Open protocol
+ Expand
4

Histochemical Analysis of Intestine

Check if the same lab product or an alternative is used in the 5 most similar protocols
The intestine was processed for histochemical analysis as described (Cox et al., 2018 (link)). Antibodies used were: Cyclin D1 (cat. 2978; Cell Signaling), E-cadherin (cat. 3195; Cell Signaling), Ki67 (cat 15580; Abcam), OLFM4 (cat. 39141; Invitrogen), Chromogranin A (Chgr A) (cat. ab15160; abcam), rabbit anti-HES1 (cat. 11988; CST) and anti-EDTB hybridoma supernatant (Wilson et al., 1987 (link)). Antigen retrieval with Tris/EDTA pH 9 was used for Cyclin D1 (1:50). Citrate pH 6 antigen retrieval was used for E-cadherin (1:200), Ki67 (1:200), Chgr A (1:200), HES1 (1:200) and OLFM4 (1:400). Signal Boost (cat. 8114S; Cell Signaling Technology) and DAB detection kits (cat. 8059S; Cell Signaling Technology) were used for visualization of Cyclin D1 and HES1. To visualize goblet cells, tissue was labelled with Alcian Blue Solution according to manufacturer’s instructions (cat. IW-3000A; IHCWORLD).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!