For detection of Lgr5 and Olfm4 mRNA, we designed riboprobes targeting the coding sequence to 3’UTR region of these genes (Supplemental Table 5 ). Targeted probe region DNA were amplified using standard PCR, with the amplicon length of about 500 bp. These DNA amplicons were used to generate DIG-labeled riboprobes using T7 RNA polymerase. For staining, Formalin fixed paraffin embedded tissue sections were deparaffinized with Citrisolv, washed in 100% ethanol, and air dried. Slides were blocked with Boeringer Blocking Reagent (Roche) and yeast tRNA at 68°C for 1 hour. 0.5 ng/mL probes were hybridized to sections at 68°C overnight. After washing, sections were again blocked with Boeringer Blocking Reagent and 10% sheep serum at room temperature for 1 hour. Alkaline phosphatase labeled sheep anti-DIG antibody (Roche) was incubated with sections at 1:2000 dilution at 4°C overnight. After washing, positive signals were developed using NBT/BCIP substrate (Thermo). Slides were then fixed in 10% neutral buffered formalin and mounted with aqueous mounting medium.
Boeringer blocking reagent
Manufactured by Roche
Boeringer Blocking Reagent is a laboratory product used to block non-specific binding sites in immunoassays. It is a protein-based solution designed to reduce background signal and improve the specificity of immunological detection methods.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using boeringer blocking reagent
1
In Situ Hybridization of Lgr5 and Olfm4
2
In Situ Hybridization of Lgr5 and Olfm4
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For detection of Lgr5 and Olfm4 mRNA, we designed riboprobes targeting the coding sequence to 3’UTR region of these genes (Supplemental Table 5 ). Targeted probe region DNA were amplified using standard PCR, with the amplicon length of about 500 bp. These DNA amplicons were used to generate DIG-labeled riboprobes using T7 RNA polymerase. For staining, Formalin fixed paraffin embedded tissue sections were deparaffinized with Citrisolv, washed in 100% ethanol, and air dried. Slides were blocked with Boeringer Blocking Reagent (Roche) and yeast tRNA at 68°C for 1 hour. 0.5 ng/mL probes were hybridized to sections at 68°C overnight. After washing, sections were again blocked with Boeringer Blocking Reagent and 10% sheep serum at room temperature for 1 hour. Alkaline phosphatase labeled sheep anti-DIG antibody (Roche) was incubated with sections at 1:2000 dilution at 4°C overnight. After washing, positive signals were developed using NBT/BCIP substrate (Thermo). Slides were then fixed in 10% neutral buffered formalin and mounted with aqueous mounting medium.
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