In-place hybridization experiment requirements and procedures.

In situ hybridization
tissue
(or cell) chemistry (In Situ Hybridization, ISHH) is a method of detecting specific nucleic acid sequences in tissue cells in situ by using labeled
DNA
or RNA as a probe.
, depending on the probe used and the target nucleic acid, in-place hybridization can be divided into DNA-DNA hybridization, DNA-RNA hybridization and RNA-RNA hybridization. Depending on whether the marker of the probe is detected directly, in-place hybridization can be divided into direct and indirect methods.
direct method mainly uses radioactive
isotopes
, fluorescence and some enzyme-labeled probes and target nucleic acids for hybridization, after hybridization through radiation self-development, fluorescence
scope
or chromase-escase-thromatization reactions directly displayed. The inter-connection method generally marks the probe with half
antigen
and finally shows the hybrid formed between the probe and the target nucleic acid indirectly by the immunobiobiic chemistry method against the semi-anti-original position.
in-place hybridization was originally carried out with isotope-labeled probes. Although isotope markers (e.g. 35S, 3H, 32P, etc.) are still widely used, the rapid development of non-isotope marker probes (especially biotin marker probes and geosoxin marker probes) is of great interest to scientists.
, the basic requirements of
the organization: the organization should be as fresh as possible. Because tissue RNA degrades faster, fresh tissue and
culture
cells are best fixed within 30min.
are:
(1) to maintain cellular structure;
(2) to maximize the level of DNA or RNA in cells; and
(3) to make it easy for probes to enter cells or tissues.
's most commonly used fixant is polyformaldehyde, which, unlike other aldehyde fixants such as dialdehyde, does not produce a wide cross-connection with
proteins
and does not affect the probe penetration into cells or tissues.
Enhances the penetration of tissue permeability and nucleic acid probe:
(1) thin acid treatment and acidic acid treatment: to prevent static binding between the probe and alkaline proteins in the tissue to reduce the background, pre-hybrid specimens can be treated with 0.25% acetic anhydride 10min, after acetic acid treatment, the alkaline group in the tissue protein is blocked by acetylation. Tissue and cell specimens can also be treated with 0.2M HCl 10min, thin acid can make alkaline protein denaturation, combined with protease digestion, easy to remove alkaline protein.
(2) detergent treatment: The purpose of detergent treatment is to increase the permeability of the tissue, which is good for hybrid probes into tissue cells, and the most commonly used detergent is Triton X-100. Note: Excessive detergent treatment not only affects the morphological structure of the tissue, but also causes the loss of target nucleic acids.
(3) Protease treatment: Protease digestion can expose the target nucleic acids that are blocked after fixation to increase access to the target nucleic acids. Commonly used proteases include proteinase K, as well as streptomycin (pronase) and gastric protease (pepsin).
Hybrid buffer incubation:
Incubate 2hr at hybrid temperature with a probe-free hybrid buffer before hybridization to block points in slides and specimens that may produce nonse specific binding to the probe for the purpose of reducing the background.
Preventing contamination:
Because RNA enzymes may be found in finger skin and laboratory glassware, disinfection
gloves
are required throughout the prebreeding process to prevent contamination from affecting the results of the experiment, and glassware and tweezers used in the experiment should be baked at high temperatures (180 degrees C) the day before the experiment to eliminate RNA enzymes. Solutions used before and during hybridization are subject to high-pressure disinfection.
dual-stranded DNA probes and target DNA denaturation:
hybridization, both the probe and the target nucleic acid must be single-stranded. If hybridization is carried out with a double-stranded DNA probe (including when RNA is detected), the double-stranded DNA probe must be denatured before hybridization. The probe denaturation should be carried out immediately after the hybrid reaction, otherwise the probe of the unchained will be re-re-reseeded.
.