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Chemical staining method using niacinamide adenine dinucleotide diaphorase tissue

A nicotinamide adenine and dinucleotide technology, applied in the field of biological staining, can solve the problems of increasing operation steps and time, not recommending the diagnosis of megacolon, lack of specificity of nerve fibers, etc., to achieve the effect of simple process

Inactive Publication Date: 2014-12-10
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been reported that the combination of HE method and AchE staining can better diagnose HD, but it increases a lot of operation steps and time, which is not conducive to rapid intraoperative diagnosis
As for the LDH staining method, because it can form blue-purple granular deposits in ganglion cells, sometimes the range of intestinal segments without ganglion cells can be judged, but this enzyme lacks specificity for ganglion cells and nerve fibers. Moreover, AchE staining is generally used in combination, so it is not recommended for the diagnosis of megacolon

Method used

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  • Chemical staining method using niacinamide adenine dinucleotide diaphorase tissue
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  • Chemical staining method using niacinamide adenine dinucleotide diaphorase tissue

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Materials and Methods:

[0020] 1) Specimens and grouping The specimens used in this study were all obtained from intestinal resection during operation, and were approved by the ethics committee. From 2008 to 2011, 37 HD children underwent intraoperative colon resection, including 27 males and 10 females, aged 5 to 19 months, with an average of 11.6 months. Divided into dilation segment, shifting segment, and stenosis segment, they were stored in ultra-low temperature refrigerators in batches after production, and non-HD colostomy resection specimens were used as positive controls. Divided into NADPH-d rapid staining group, frozen section HE staining group.

[0021] 2) The specimens were routinely frozen and serially sectioned, with a thickness of 4 μm, mounted on a poly-lysine slide, and the odd-numbered sections were taken for NADPH-d rapid histological staining, and the even-numbered sections were used for HE staining of frozen sections.

[0022] 3) Rapid histologi...

Embodiment 2

[0034] Materials and Methods:

[0035]1) Specimen source The specimens were all taken from the colons removed during radical resection of Hirschsprung’s colon in 18 HD children in our hospital from January 2009 to December 2012. There were 11 males and 7 females, ranging in age from 3 to 16 years. months, with an average of (8.6±3) months. The specimens were divided into dilation section, shifting section and stenosis section. The transverse section of the intestinal tube was routinely frozen sectioned, with a thickness of 4 μm, and poly-lysine slides were pasted. Random odd-numbered slices were quickly stained with NADPH-d method, and even-numbered slices were stained with Ache method. The specimens used were approved by the Hospital Ethics Committee.

[0036] 2) Improved NADPH-d enzyme histochemical reaction: Add 0.5 mg NADPHd (Sigma), 10 μL dimethyl sulfoxide (Suzhou Zhengxing Chemical Research Institute), 0.6 mg NBT (Sigma), 3 μL Triton X-i00 (Beijing Baierdi biological...

Embodiment 3

[0051] Materials and Methods:

[0052] 1) The specimens used were approved by the Hospital Ethics Committee, and were all obtained from intestinal resection during operation. From 2009 to 2012, 18 HD children underwent intraoperative colon resection, including 11 males and 7 females, aged 3 to 16 months. Dilated segment, shifted segment, and stenotic segment were divided into slices and stored in ultra-low temperature refrigerators in batches after production. Colostomy resection specimens from non-HD children were used as positive controls. Divided into reduced nicotinamide adenine dinucleotide dehydrogenase (NADPH-d) staining group, HE staining group, acetylcholinesterase (AchE) staining group.

[0053] 2) Preparation of slides: The specimens were routinely frozen and serially sectioned, with a thickness of 4 μm, mounted on poly-lysine glass slides. The odd-numbered sections were randomly selected for NADPH-d rapid histological staining, and the even-numbered sections were ...

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Abstract

The invention relates to a chemical staining method using a niacinamide adenine dinucleotide diaphorase tissue. The method comprises the following steps: conventionally freezing and serially sectioning a production sample, dropwise adding a reaction solution, and putting a glass slide in a water bath for 10min at the temperature of 37 DEG C; observing that the reaction solution is changed from yellow to lavender; observing ganglion cells on the taken out glass slide through a light microscope, clearly displaying, performing PBS washing, and ending the reaction; and straining for 10s through eosin, performing the PBS washing, sealing the glass slide through a glass slide sealing reagent-Clear mount, re-sealing the glass slide through neutral gum after the glass slide is completely dried, after the quick histological straining for dual-straining NADPH-d is completed, washing for 5min through tap water, acquiring images, and performing statistical treatment on all data through software. According to the technical scheme, the method is simple in flow, hydration, dehydration and other flows are not required, the reaction only needs to be performed at the temperature of 37 DEG C, and a result can be obtained within 5-10min.

Description

technical field [0001] The invention belongs to the technical field of biological dyeing, in particular to a chemical dyeing method of reduced nicotinamide adenine dinucleotide diaphorase. Background technique [0002] Hirschsprung's disease (HD) is a common developmental malformation of the digestive tract in children, which can cause intestinal obstruction and chronic constipation in infants. The incidence rate in my country is about 1:5000, and it is one of the most common congenital malformations in pediatric surgery. Generally speaking, the scope of the lesion segment is judged based on the experience of the surgeon and the general observation of the bowel during surgery. If HE staining of a frozen section is performed during the operation, atypical cases or insufficient experience of pathologists will obviously affect the bowel resection. The determination of the scope of the disease resulted in poor surgical results, and some children had recurrent constipation or fecal...

Claims

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Application Information

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IPC IPC(8): G01N1/30
Inventor 朱利斌李仲荣陈肖鸣张普夏立广王永飚谢小志
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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